Monocins and methods of use

ABSTRACT

The disclosure relates to the identification, cloning, and expression of a genetic locus within a  Listeria monocytogenes  genome that encodes a phage tail-like bacteriocin (PTLB), termed a monocin. Also provided are non-natural monocins, which have been engineered to have altered bactericidal specificity. Nucleic acid molecules encoding natural or non-natural monocins, vector constructs containing such nucleic acids operably linked to a heterologous promoter, producer cells containing such vectors, the encoded monocins, as well as methods of making and using such monocins are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States ProvisionalApplication Nos. 62/076,691, filed Nov. 7, 2014, and 62/245,493, filedOct. 23, 2015, the contents of all of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

This application relates generally to the identification, isolation,modification and expression of a cluster of genes sufficient to producea bacteriocin, and more specifically, a Phage tail-like bacteriocin(PTLB) that specifically kills Listeria species, and methods to alterits bactericidal specificity, produce, and use the same.

Background Information

Listeria is a genus of bacteria, which includes at least fifteenspecies. Listeria species are gram-positive bacilli that are facultativeanaerobes (i.e., capable of surviving in the presence or absence ofoxygen). The major human pathogen in the Listeria genus is L.monocytogenes, which can grow and reproduce inside the infected host'scells and is one of the most virulent food-borne pathogens. L.monocytogenes is usually the causative agent of the relatively rarebacterial disease, listeriosis. Listeriosis is a serious disease forhumans caused by eating food contaminated with the bacteria. The diseaseaffects primarily pregnant women, newborns, adults with weakened immunesystems, and the elderly. The overt form of the disease has a mortalityrate of about 20 percent. The two main clinical manifestations aresepsis and meningitis. Listeria ivanovii is a pathogen of mammals,specifically ruminants, and has rarely caused listeriosis in humans.

Several strains of Listeria sps (monocytogenes, innocua, ivanovii) havebeen shown upon induction of the SOS response to produce high molecularweight (HMW) bacteriocins or Phage tail-like bacteriocins (PTLBs) termed“monocins” (Zink et al., 1994). These particles are released into themedium upon lysis of the monocin producer cells and have been shown byspot plate assay to have bactericidal activity on other Listeriastrains. Particle production was confirmed by electron microscopy.Monocins produced by different strains displayed different bactericidalspectra. The genetic locus encoding a monocin has not been identified.The sequence of a putative monocin lytic enzyme was erroneouslydescribed many years ago (Zink et al., 1995; see below).

SUMMARY OF THE INVENTION

The present invention relates to the identification, cloning, andexpression of a genetic locus within a Listeria genome that as a clusterof genes encodes a Phage tail-like bacteriocin (PTLB), termed a monocinor listeriocin, interchangeably. The present invention also relates tomodified monocins. Monocins contain a receptor binding protein (RBP)that directs the binding of the monocin to the bacterium that it kills.

Accordingly, in one aspect, there are provided isolated nucleic acidmolecules encoding a non-natural monocin, wherein the nucleic acidmolecule contains a first polynucleotide that encodes a monocinstructural scaffold, and a second polynucleotide encoding a heterologousRBP, wherein the scaffold contains all structural proteins of afunctional monocin except its corresponding natural RBP, and wherein themonocin has bactericidal specificity as determined by the heterologousRBP. In some embodiments, the scaffold encoded by the firstpolynucleotide is at least 80% identical to SEQ ID NOs: 7-16, amino acidsequences ORFs 130-139.

In another aspect, there are provided producer cell integration vectorscontaining the disclosed nucleic acid molecule encoding a monocin,wherein the nucleic acid molecule is operably linked to a heterologousinducible promoter. In some embodiments, the producer cell is Bacillussubtilis. B. subtilis does not naturally produce a monocin.

In still another aspect, there are provided nucleic acid moleculesencoding a monocin, wherein the nucleic acid molecule contains apolynucleotide that encodes amino acid sequences that are at least 80%identical to SEQ ID NOs: 5-17 and a heterologous promoter inducible by asmall molecule, wherein the monocin has bactericidal activity, andwherein the polynucleotide is operably linked to the heterologouspromoter. In particular embodiments, the promoter is placed atapproximately 11, 14, 17, 20, or 23 nucleotides upstream of the portionof the polynucleotide encoding SEQ ID NO: 5. In a further aspect, thereare provided monocin producer cells containing the disclosed nucleicacid molecules encoding a monocin. In some embodiments, the monocinproducer cell contains a first foreign polynucleotide that encodes aminoacid sequences that are at least 80% identical to SEQ ID NOs: 7-16 and asecond foreign polynucleotide encoding an RBP, wherein the first andsecond polynucleotides encode a monocin having bactericidal specificityas determined by the RBP. In yet another aspect, there are providedmethods of producing a monocin, by exposing a monocin producer cellcontaining a nucleic acid molecule encoding a monocin, wherein thenucleic acid molecule is operably linked to a heterologous induciblepromoter, to an inducing agent in a concentration effective to induceexpression of the monocin via the inducible promoter, thereby producingthe monocin. In some embodiments, the nucleic acid molecule encoding amonocin is integrated within the genome of the producer cell in order togenerate a stable monocin producer cell. In another aspect, there areprovided methods of killing a Listeria species, comprising contactingthe Listeria species with an effective amount of a monocin of thedisclosure, whereby the monocin binds and kills the Listeria species. Insome embodiments, the contacting is with a surface contaminated withListeria species. In one example, the contacting is at 2-10° C. Inanother aspect, there are provided methods of treating an infection ofListeria species in an animal comprising, administering to an animal inneed thereof an amount of a monocin of the disclosure, or a monocinproducer cell of the disclosure in an amount sufficient to produce abactericidal amount of the monocin, thereby treating the Listeriainfection or colonization.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Map of the genetic locus of monocins. The top is the entire wildtype locus including regulatory, structural, and lysis genes asindicated. A. The structural genes of the natural monocin which encodethe scaffold and the natural RBP are shown. B-D show examples ofnon-natural monocins with 3 representative types of heterologous RBPs.B. A non-natural monocin with a mutant or modified native RBP. C. Anon-natural monocin with an unmodified native but heterologous RBP (anexample is monocin 35152-33090 of this invention). D. A non-naturalmonocin with an RBP fusion in which an amino-terminal portion of themonocin BPAR is fused to a heterologous RBD, which can be derived from abacteriophage, prophage, or prophage remnant (an example is monocin35152-A118 of this invention).

FIG. 2. Spot assay of the monocin 35152-33090 produced in B. subtilisstrain sGL-075. This non-natural monocin gene cluster is undertranscriptional control of the Phyper-spank promoter. The targetbacterium is L. monocytogenes strain 19111.

FIG. 3. Bactericidal spectrum chart of the activities of naturalmonocins 35152 and 33090 and non-natural monocins 35152-33090 and35152-A118 against the indicated target strains. Shaded squares indicatebactericidal killing. Clear squares indicate insensitivity.

FIG. 4. The results of assays showing the bactericidal activities ofmonocins at 3-4° C. Lawns of target L. monocytogenes bacteria werechilled to 3-4° C. and spotted with chilled preparations of monocins andthen incubated at 3-4° C. for 3 days before imaging. Lane A is L.monocytogenes strain 23074 spotted with monocin 35152 and Lane B is L.monocytogenes strain 15313 spotted with monocin 35152-A118. Clearing inthe lawn indicated bactericidal activity.

FIG. 5. Spot tests of monocin 35152-A118 produced in constructsincluding various combinations of putative tail fiber assembly orchaperone genes. If no putative tail fiber genes were present, no activemonocin was produced. The inclusion of ORF21 resulted in robust activitywhich was equal to the activity of the monocins produced with both ORFs21 and 22. Having all three putative tail fiber assembly proteins wasactually detrimental.

FIG. 6. Comparison on monocins produced in the Δ8 strain vs BDG9.

FIG. 7. Comparison of the monocin gene cluster to the genome of theTP901-1-like phage A118. The genes that are similar are colored black.Three regulatory genes of the monocin cluster (0126, 0128, and 0129)were homologues of regulatory genes of phage A118. Five of the monocinmajor structure genes (0131, 0134, 0135, 0136, and 0140) were also A118homologues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification, cloning, andexpression of a genetic locus within a Listeria genome that encodes aPhage tail-like bacteriocin (PTLB), termed a monocin or listeriocin.Also provided are modified or non-natural monocins, such as those thathave been engineered to have altered bactericidal specificity.Accordingly, provided herein are nucleic acid molecules encoding naturalor non-natural monocins, integration vector constructs containing suchnucleic acids operably linked to a heterologous promoter, producer cellsthat do not naturally produce monocins but containing such nucleic acidmolecules or vectors, the encoded monocins, as well as methods of makingand using such monocins.

As used interchangeably herein, the terms “Phage tail-like bacteriocin”(PTLB) and high molecular weight (HMW) bacteriocin may include, F-typebacteriocins (FTBs) and R-type bacteriocins (RTBs). For example, amonocin is a PTLB. The present inventors previously posited thatmonocins as contemplated herein have the structures of (RTBs), see U.S.Provisional Application No. 62/076,691, but as described herein, moreclosely resemble FTBs. A PTLB may be natural or non-natural, that is itexists in nature or does not exist in nature, respectively.

The terms “monocin” and “listeriocin” are used interchangeably herein,and refer to a PTLB isolated from or derived from a Listeria species.Monocins disclosed herein are complex molecules comprising multipleprotein, or polypeptide, subunits and distantly resemble the tailstructures of bacteriophages. In naturally occurring monocins thesubunit structures are encoded by a genetic locus present within thebacterial genome such as that of L. monocytogenes, L. ivanovii, or L.innocua, and form monocins to serve as natural defenses against otherbacteria. Monocins may be natural or non-natural.

A functional monocin contains a structural scaffold and an RBP (see FIG.1A). Thus, the “structural scaffold” (used interchangeably with “monocinstructural scaffold” or “scaffold”) contains all of the structuralproteins of a functional monocin except the RBP. In some embodiments,the scaffold includes the open reading frames (ORFs) corresponding toORFs 130-139 of Listeria strain 35152. In particular embodiments, thestructural scaffold includes SEQ ID NOs: 7-16. In other embodiments, thestructural scaffold is at least 80% identical to SEQ ID NOs: 7-16. Inother embodiments, the structural scaffold has an amino acid sequencethat is at least 85%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or even 99%identical to a polypeptide containing ORFs 130-139 or SEQ ID NOs: 7-16.

The RBP consists of an amino terminal portion that provides attachment(termed the “baseplate attachment region” or BPAR) to the rest of themonocin structural scaffold and a carboxy terminal portion that providesa receptor binding domain (RBD) that is the targeting motif of the RBP.In some embodiments, the BPAR is natural to the structural scaffold. Inother embodiments the BPAR is highly homologous to the BPAR native tothe structural scaffold. In particular embodiments, the BPAR is at least80% identical to the BPAR native to the structural scaffold. Inparticular embodiments, the BPAR includes only the amino terminal 20 to60 amino acids of ORF 140 (see FIG. 1D).

“Natural monocins” as used herein refer to those monocins that exist innature, and include native particles obtained from Listeria, as well asparticles obtained through expression of a natural monocin gene clusterin a monocin producer cell that does not in nature produce a monocin(see FIG. 1A).

“Non-natural monocins” as used herein refer to those monocins that donot exist in nature (see FIG. 1B-1D). In other embodiments, thenon-natural monocin contains a heterologous RBP. A “heterologous RBP”may be a native RBP obtained from a different source than was thestructural scaffold to which it is attached (see FIG. 1B); or aheterologous RBP may be a modified RBP that was a natural RBP prior tobeing modified or mutated to change its physical and/or biologicproperties (see FIG. 1C). In some embodiments, a modified RBP is onethat contains an amino acid sequence that is different (e.g., engineeredto differ) from a native or natural RBP and confers to the resultingnon-natural monocin different receptor binding properties (see FIG. 1C).In other embodiments, a modified RBP may be comprised of a fusionbetween an amino terminal portion of a natural RBP (the BPAR) and aheterologous Receptor Binding Domain (RBD) (see FIG. 1D). An RBD is thatportion of an RBP that directs the RBP, which, in turn, directs the PTLBto its specific target bacteria. In one example, a non-natural monocinmay be an engineered PTLB particle comprised of polypeptides encoded bygenes derived from one or more strains of Listeria species and 80% ormore identical to the structural proteins encoded by SEQ ID NOs: 7-16that, when incorporating a heterologous RBP, makes up a complete, activemonocin.

Accordingly, there are provided nucleic acid molecules encoding anon-natural monocin, wherein the nucleic acid molecule includes a firstpolynucleotide that encodes all structural proteins of a functionalmonocin except a corresponding natural RBP, wherein the nucleic acidmolecule further includes a heterologous second polynucleotide sequenceencoding the heterologous RBP, and wherein the non-natural monocin hasbactericidal specificity as determined by the heterologous RBP. Inparticular embodiments, the structural proteins encoded by the firstpolynucleotide correspond to ORFs 130-139 of a monocin genetic locus. Inone example, the structural proteins are SEQ ID NOs: 7-16.

In particular embodiments, a non-natural monocin may include an RBPfusion. In one such example, the non-natural monocin contains astructural scaffold and an RBP fusion consisting of the BPAR from thecorresponding natural RBP and a heterologous RBD. In some examples, theBPAR includes amino acid positions 1-40 of the natural RBP. To make acomplete non-natural monocin molecule, the RBP fusion is attached to thestructural scaffold, whereby the heterologous RBD determines thebactericidal spectrum of the resulting non-natural monocin. In exampleswhere the non-natural monocin contains a heterologous RBP which is anRBP fusion, the nucleic acid molecule encoding the scaffold and theheterologous RBP is engineered so that the resulting monocin willcontain a heterologous RBP consisting of amino acids at positionsapproximately 1-40 of the natural BPAR fused to the carboxy terminalportion of a heterologous RBD (see FIG. 1D).

As used herein, a “nucleic acid” or a “nucleic acid molecule” typicallyrefers to deoxyribonucleotide or ribonucleotide polymers (pure or mixed)in single- or double-stranded form. The term may encompass nucleic acidscontaining nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,which have similar binding, structural, or functional properties as thereference nucleic acid, and which are processed in a manner similar tothe reference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-0-methyl ribonucleotides, andpeptide-nucleic acids (PNAs). The term nucleic acid may, in somecontexts, be used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also encompasses conservativelymodified variants thereof (such as degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third (“wobble”) position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues. Thus, a nucleic acid sequence encoding a proteinsequence disclosed herein also encompasses modified variants thereof asdescribed herein. The terms “polypeptide”, “peptide”, and “protein” aretypically used interchangeably herein to refer to a polymer of aminoacid residues. Amino acids may be referred to herein by either theircommonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The term “segment” as used herein in reference to an amino acid sequencerefers to a contiguous sequence of amino acids that may be 10, 12, 15,20, 25, 50, or 100 amino acid residues in length. As used herein, theterm “heterologous,” when used with reference to portions of a proteinor nucleic acid sequence, indicates that the sequence comprises two ormore subsequences that are not usually found in nature in the samerelationship to each other. In one example, the heterologous sequencesare from different species of bacteria. In another example, heterologoussequences are from different strains of the same species of bacteria. Inone aspect, the heterologous sequences are from different strains of L.monocytogenes. In another aspect the heterologous sequences are from abacterium and a bacteriophage or prophage, or from a bacterium and asynthetic, non-natural sequence of DNA.

The heterologous RBP may be comprised of an RBD obtained from anotherstrain of L. monocytogenes, another species of Listeria, or a genus ofbacteria other than the species and strain of the bacteria from whichthe scaffold was derived. In some embodiments, the species of Listeriainclude L. fleischmannii, L. grayi, L. innocua, L. ivanovii, L. marthii,L. rocourtiae, L. seeligeri, L. weihenstephanensis and L. welshimeri. Inother embodiments, the genus of bacteria is selected from Clostridium,Staphylococcus, Streptococcus, Bacillus, Enterococcus,Propionibacterium. In some embodiments, the heterologous RBD is from aL. monocytogenes genome, a bacteriophage, a prophage insertion or aprophage remnant that is contained within a Listeria genome. A “prophageremnant” or prophage element or portion, refers to a sequence thatencodes only a portion of a phage or discrete phage protein(s), ratherthan a full phage structure. Thus, in some embodiments, a prophageremnant may include, for example, sequence encoding an RBD and otherstructural proteins. In certain embodiments, the RBD is of a prophage orprophage remnant from the genome of a gram positive bacterium or an RBDof a bacteriophage that infects a gram positive bacterium. In oneexample, the gram positive bacterium is a species of Clostridium,Staphylococcus, Streptococcus, Bacillus, Enterococcus, orPropionibacterium. In some embodiments, the natural RBP of a naturalmonocin may be replaced with a modified form of a native RBP. A “nativeRBP” refers to a RBP having an amino acid sequence that is identical toa RBP isolated or cloned from another strain of L. monocytogenes or froma bacteriophage that infects L. monocytogenes or from another genus orspecies of bacteria or from a bacteriophage. Exemplary native RBP fromL. monocytogenes include SEQ ID NOs: 17 and 26, from strains 35152 andF6854, respectively, and an exemplary native RBP from L. innocuaincludes SEQ ID NO: 27 from strain 33090. In some embodiments, amodified RBP includes a change in the amino acid sequence of the RBPrelative to a native RBP. Non-limiting examples of a change in aminoacid sequence include substitution, insertion (or addition), or deletionof one or more amino acids that modifies the binding or stabilityproperties of the RBP.

In particular embodiments, the modified form of a native RBP alsoresults in a monocin having a heterologous RBP and bactericidal spectrumthat is different from a monocin containing the corresponding unmodifiedor native RBP. In particular embodiments, the modified form is at least80% identical to the native RBP. In other embodiments, the RBP has anamino acid sequence that is at least 85%, 88%, 89%, 90%, 95%, 96%, 97%,98%, or even 99% identical to a polypeptide selected from the groupconsisting of SEQ ID NOs: 17, 26, 27 and the modified RBP results in amonocin having a bactericidal spectrum that is different from a monocinhaving the corresponding unmodified or native RBP.

Also provided are variant monocins. Variant monocins include thosemonocins having an amino acid sequence that is at least 80% identical toa polypeptide containing ORFs 130-139 (SEQ ID NOs: 7-16), or ORFs130-140 (SEQ ID NOs: 7-17). In other embodiments, the variant monocinhas an amino acid sequence that is at least 85%, 88%, 89%, 90%, 95%,96%, 97%, 98%, or even 99% identical to a polypeptide containing ORFs130-139, or ORFs 130-140.

Also provided are vectors or expression constructs containing a nucleicacid molecule encoding a monocin. In some embodiments, the nucleic acidmolecule is operably linked to a heterologous inducible promoter in thevector or expression construct. In particular embodiments, theheterologous promoter is a small molecule induced promoter. Examples ofsuch small molecule induced promoters include P_(LAC) (lactose, IPTG),P_(TAC) (IPTG), P_(BAD) (arabinose), and P_(XYL) (Xylose). In particularembodiments, the promoter is placed at approximately 17 nucleotidesupstream of a polynucleotide encoding ORF 128 (SEQ ID NO: 5) of themonocin.

In other embodiments, the vector or expression construct may include oneor more regulatory proteins encoded by a monocin genetic locus or genecluster. In particular embodiments, the one or more regulatory proteinsare encoded by an ORF selected from the group consisting of ORFs 125,126, 127, 128, and 129 (SEQ ID NOs: 2-6, respectively). In one example,the one or more regulatory proteins are encoded by an ORF selected fromthe group consisting of SEQ ID NOs: 2-6.

A monocin of the invention may be cold active, that is, it hasbactericidal activity in cold temperatures, such as 2-10° C.

An additional property common to the monocins disclosed herein is thatthey do not contain nucleic acid and thus, are replication deficientsuch that they cannot reproduce themselves after or during the killingof a target bacterium, as can many bacteriophages. They are purelyproteins, not organisms or viruses.

A “target bacterium” or “target bacteria” refers to a bacterium orbacteria that are bound by a monocin of the disclosure and/or whosegrowth, survival, or replication is inhibited thereby. In someembodiments, the target bacterium is from the genus Listeria. In someembodiments, the target bacterium is from a species of Listeria selectedfrom the group consisting of L. monocytogenes, L. innocua, and L.ivanovii. In particular embodiments, the bacterium is Listeriamonocytogenes. In one aspect, more than one strain of L. monocytogenesis targeted. Exemplary strains of Listeria monocytogenes include, butare not limited to, strain 15313 (serovar 1/2a), strain 19111 (serovar1/2a), strain 35152 (serovar 1/2a), strain DD1144 (serovar 1/2a), strainDD1145 (serovar 1/2a), strain DD1152 (serovar 1/2a), strain DD1299(serovar 1/2a), strain DD1313 (serovar 4b), strain DD1294 (serovar 4b),strain DP-L4056 (serovar (1/2a), strain DP-L3633 (serovar 1/2a), strainDP-L3293 (serovar 1/2c), strain DP-L3817 (serovar 1/2a), strain DP-L1171(serovar 1/2b), strain DP-L185 (serovar 4b), strain DP-L186 (serovar4b), strain DP-L188 (serovar 3), strain DP-L1173 (serovar 4b), strainDP-L1174 (serovar 4b), strain DP-L1168 (serovar 4b), strain DP-L1169(serovar 4b), strain 23074 (serovar 4b), and Listeria ivanovii strain19119 (serovar 5). In some embodiments, the target bacterium is from thegenus Clostridum, Staphylococcus, Streptococcus, Bacillus, Enterococcus,or Propionibacterium. The term “growth inhibition” or variations thereofrefers to the slowing or stopping of the rate of a bacterial cell'sdivision or cessation of bacterial cell division, or to the death of thebacterium or bacteria.

Virulence factors are those molecules that contribute to thepathogenicity of an organism but not necessarily its general viability.Upon the loss of a virulence factor the organism is less pathogenic to ahost but not necessarily less viable in culture. Virulence factors mayhave any one of numerous functions, for example, regulating geneexpression, providing adhesion or mobility, providing a toxin, injectinga toxin, pumping out antibiotic agents, or forming protective coatingsincluding biofilms.

Fitness factors are those molecules that contribute to the organism'sgeneral viability, growth rate or competitiveness in its environment.Upon the loss of a fitness factor, the organism is less viable orcompetitive and because of this compromise, indirectly less pathogenic.Fitness factors may also possess any one of numerous functions, forexample, acquiring nutrients, ions or water, forming components orprotectants of cell membranes or cell walls, replicating, repairing ormutagenizing nucleic acids, providing defense from or offense towardsenvironmental or competitive insults.

Monocins targeting surface accessible virulence or fitness factors(e.g., Internalins on the surfaces of Listeria species and S-layerproteins, prevalent on many bacteria, the Clostridium species, forexample) offer an attractive means of forcing such pathogens tocompromise their virulence or fitness if they emerge as resistant to themonocin.

In additional embodiments, a monocin as provided herein is used to treatfood or food storage areas contaminated with target bacteria. Inparticular embodiments, the monocin is cold stable, cold active, and isused to treat bacterial contamination of refrigerated food orrefrigerated storage areas. Accordingly, there are provided methods ofkilling Listeria monocytogenes by contacting the L. monocytogenes withan effective amount of a monocin, whereby the monocin binds and killsthe L. monocytogenes. In some embodiments, the contacting is in ananimal and a bactericidal amount of the monocin is administered to theanimal. In other embodiments, the contacting is with a surfacecontaminated with L. monocytogenes. In certain embodiments, thecontacting is in the cold, for example at 2-10° C.

Also provided, are methods of treating an infection of L. monocytogenesin an animal by administering to an animal in need thereof an amount ofa monocin, or a monocin producer cell to produce a bactericidal amountof the bacteriocin, thereby treating the infection.

As described herein, an anti-bacterial monocin may be used to inhibitgrowth, survival, or replication of a particular bacterium. Thebacterium may be a pathogenic or environmentally deleterious strain, ormay be treated in a prophylactic manner. A pathogenic microorganismgenerally causes disease, sometimes only in particular circumstances.

An engineered monocin of the disclosure may be administered to anysubject afflicted with, diagnosed as afflicted with, or suspected ofbeing afflicted with, an infection, colonized by, or contamination bybacteria susceptible to the monocin. Non-limiting examples of such asubject include animal (mammalian, reptilian, amphibian, avian, andfish) species as well as insects, plants and fungi. Representative, andnon-limiting, examples of mammalian species include humans; non-humanprimates; agriculturally relevant species such as cattle, pigs, goats,and sheep; rodents, such as mice and rats; mammals for companionship,display, or show, such as dogs, cats, guinea pigs, rabbits, and horses;and mammals for work, such as dogs and horses. Representative, andnon-limiting, examples of avian species include chickens, ducks, geese,and birds for companionship or show, such as parrots and parakeets. Ananimal subject treated with an engineered monocin of the disclosure mayalso be a quadruped, a biped, an aquatic animal, a vertebrate, or aninvertebrate, including insects.

In some embodiments, the subject in need to be treated is a human childor fetus or other young animal which has yet to reach maturity. Thus thedisclosure includes the treatment of pediatric or obstetric conditionscomprising infection with bacteria or other microorganism susceptible toa monocin of the disclosure.

In some embodiments, there are provided compositions of more than onenon-natural monocin, wherein the non-natural monocins have differingbactericidal spectra. In other embodiments, there are providedcompositions of one or more non-natural monocins and one or more naturalmonocins, wherein the monocins have differing bactericidal spectra.

In some embodiments, monocins, combinations of monocins, or monocinproducer cells capable of producing monocins are formulated with a“pharmaceutically acceptable” excipient, enteric coating or carrier.Such a component is one that is suitable for use with humans, animals,and/or plants without undue adverse side effects. Non-limiting examplesof adverse side effects include toxicity, irritation, and/or allergicresponse. The excipient or carrier is typically one that is commensuratewith a reasonable benefit/risk ratio. Non-limiting pharmaceuticallysuitable carriers include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples include, but are not limited to,standard pharmaceutical excipients such as a phosphate buffered salinesolution, bicarbonate solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyloleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like.

Additional formulations and pharmaceutical compositions disclosed hereincomprise an isolated monocin specific for a bacterial pathogen; amixture of two, three, five, ten, or twenty or more different monocinsor producer cells capable of producing monocins that target the samebacterial pathogen; and a mixture of two, three, five, ten, or twenty ormore that target different bacterial pathogens or different strains ofthe same bacterial pathogen.

Optionally, a composition comprising a monocin or producer cell of thedisclosure may also be spray dried or lyophilized using means well knownin the art. Subsequent reconstitution and use may be practiced as knownin the field.

A monocin is typically used in an amount or concentration that is “safeand effective”, which refers to a quantity that is sufficient to producea desired therapeutic or prophylactic response without undue adverseside effects like those described above. A monocin may also be used inan amount or concentration that is “therapeutically effective”, whichrefers to an amount effective to yield a desired therapeutic response,such as, but not limited to, an amount effective to slow the rate ofbacterial cell division, or to cause cessation of bacterial celldivision, or to cause death or decrease rate of population growth of thetarget bacteria. The safe and effective amount or therapeutically orprophylactically effective amount will vary with various factors but maybe readily determined by the skilled practitioner without undueexperimentation. Non-limiting examples of factors include the particularcondition being treated, the physical condition of the subject, the typeof subject being treated, the duration of the treatment, the nature ofconcurrent therapy (if any), and the specific formulations employed.

The terms “producer cell” and “monocin producer cell” are usedinterchangeably herein and refer to a cell that is capable of producingor expressing a monocin-encoding nucleic acid molecule, and which doesnot naturally contain such a nucleic acid molecule. The producer cellmay be capable of surviving and growing in the presence of oxygen and istransformed with a vector containing a nucleic acid molecule encodingthe monocin, which may be integrated into the chromosome of the producercell or may be episomal. The producer cell may be a gram positivebacterium. In certain embodiments, the producer cell may be a bacteriumfrom the genus Bacillus, Lactobacillus, Listeria, or Lactococcus.

In some embodiments, the bacterium is a species from the genus Bacillusselected from the group consisting of subtilis, amyloliquefaciens, andmegaterium. In one aspect, the bacterium is Bacillus subtilis. In aparticular aspect, the producer cell is a B. subtilis strain that lacksthe PBSX gene cluster SpoA, Flag, etc. In other embodiments, thebacterium is a species from the genus Lactobacillus selected from thegroup consisting of acidophilus, casei, and bulgaricus. In anotherparticular embodiment the producer cell is a species of Listeria otherthan monocytogenes capable of producing or expressing a monocin-encodingnucleic acid molecule and which does not naturally contain such anucleic acid molecule. In some embodiments, a producer cell contains afirst foreign polynucleotide that encodes an amino acid sequence that isat least 80% identical to SEQ ID NOs: 7-16 and a second foreignpolynucleotide encoding a heterologous RBP, wherein the first and secondpolynucleotides encode a monocin having bactericidal specificity asdetermined by the heterologous RBP. In particular embodiments, thesecond foreign polynucleotide is heterologous to the first foreignpolynucleotide. In some embodiments, the first and secondpolynucleotides are separate nucleic acid molecules. In otherembodiments, the first and second polynucleotides are contained in onenucleic acid molecule. The following examples are intended to illustratebut not limit the invention.

The term “comprising”, which is used interchangeably with “including,”“containing,” or “characterized by,” is inclusive or open-ended languageand does not exclude additional, unrecited elements or method steps. Thephrase “consisting of” excludes any element, step, or ingredient notspecified in the claim. The phrase “consisting essentially of” limitsthe scope of a claim to the specified materials or steps and those thatdo not materially affect the basic and novel characteristics of theclaimed invention. The present disclosure contemplates embodiments ofthe invention compositions and methods corresponding to the scope ofeach of these phrases. Thus, a composition or method comprising recitedelements or steps contemplates particular embodiments in which thecomposition or method consists essentially of or consists of thoseelements or steps.

Example 1 Identification of the Monocin Genetic Locus

This example illustrates the identification of the genetic loci thatencode a monocin within a strain of Listeria monocytogenes and a strainof Listeria innocua. Listeria monocytogenes strain ATCC 35152 andListeria innocua strain ATCC 33090 were both reported to producemonocins. (Zink et al., 1994). These two strains were induced withmitomycin C, and the monocins were collected from the lysate by highspeed centrifugation at 90,000×g. Bactericidal activity was tested bythe spot method on a panel of Listeria species. The monocins from thetwo strains were found to have differing bactericidal spectra. Neithershowed bactericidal activity against the same strain from which it wasisolated. The entire purified monocin preparations were analyzed by massspectrometry (MS) to identify in the sample proteins that had similarityto components of phage tail-like structures. Although strains 35152 and33090 are not among those in which the genome sequences are known,numerous other Listeria genomes had been sequenced and were searchable.The most abundant protein in the preparation of monocin from strain35152 corresponded to gene ImaA or antigen A encoded in numerousListeria strains. Antigen A is a protein originally found to elicit animmune response in humans with Listeria infections (Gohmann et al.,1990; Schaferkordt et al., 1997). Prior to the instant invention it wasnot known that Antigen A was actually part of a monocin. The antigen Afrom strain 35152 showed identical peptide sequences to severalhomologues in known Listeria monocytogenes genomes; the sequenced genomeof L. monocytogenes strain 1/2a F6854 was chosen as a reference, and thegene (ORF) numbering system of that strain was used for this work. TheAntigen A corresponds to ORF 131 of strain 1/2a F6854. Several otherpeptide matches were noted from the MS analyses that corresponded toORFs that are encoded in nearby regions of the genome including ORFs130, 132, 135, 136, 138, and 140 (SEQ ID NOs: 7, 9, 12, 13, 15, 17,respectively). Several of these had sequence similarities to phage tailproteins (Table 1).

TABLE 1 Open reading frames, predicted amino acid lengths, andannotations of the proteins encoded by the ORFs of the monocin genecluster. GenBank ® annotations correlate with L. monocytogenes strainF6854. AvidBiotics annotations are based on additional bioinformaticssearches, mass spectrometry, and experimental data. Length GenBank ® ORF(a.a.) Annotation AvidBiotics Annotation 125 231 HypotheticalTranscriptional regulator 126 150 Hypothetical Tox/Antitox, GP35 ofphage A118 127 117 Putative Repressor Tox/Antitox, lambda C1, HTH 128142 Antigen D Antigen D (unknown function) 129 138 Antigen CTranscriptional regulator 130 129 Antigen B Antigen B (unknown function)131 170 Antigen A Major monocin structural protein 132 100 HypotheticalMonocin structural protein 133 111 Hypothetical Monocin structuralprotein 134 622 Putative membrane Tape measure protein protein 135 272Hypothetical Monocin structural protein 136 378 Phage structural Monocinstructural protein 137  99 Hypothetical Monocin structural protein 138191 Hypothetical Monocin structural protein 139 159 Hypothetical Monocinstructural protein 140 178 Hypothetical Monocin RBP 141 140 Holin Holin142 242 N-acetylmuramoyl- Lysin L-alanine amidase

A close inspection of this genomic region revealed that ORFs 130-140(SEQ ID NOs: 7-17, respectively) encoded the components of a contractiletail structure module (FIG. 1 top). In particular, ORF 134 encoded aputative tape measure protein, and ORFs 137 and 139 encoded putativeproteins that shared at least some sequence similarity to known phagetail proteins. These ORFs are all transcribed on one strand with littleintergenic space. Just downstream, ORFs 141 and 142 (SEQ ID NOs: 18-19)encoded putative holin and lysin, the proteins that are responsible fortimed cell lysis for monocin release from the bacteria. Upstream of ORF130 there were annotated 5 putative regulatory genes. ORF 125 (SEQ IDNO: 2) had sequence similarity to transcriptional regulators; ORF 126(SEQ ID NO: 3) and 127 (SEQ ID NO: 4) had sequence similarity to phageregulatory proteins, and ORF 128 (SEQ ID NO: 5) and ORF 129 (SEQ ID NO:6) had sequence similarities to antigen D and antigen C, respectively,with ORF 129 also having some similarity to transcriptional regulators(Table 1). ORFs 125-127 were found to be transcribed in the oppositedirection from the structural genes while ORFs128 and 129 weretranscribed in the same direction as the structural genes with a gap of286 nucleotides between the end of ORF129 and the start of ORF130. A mapof the entire monocin gene cluster is shown in FIG. 1 (top). No genesencoding phage capsid, capsid assembly, or portal proteins were found inthis region or nearby in the genome. Also not present were any genesencoding DNA replication or packaging machinery, integration/excisionproteins, or any other ORFs often associated with lysogenic prophages.Thus, this region was consistent with its encoding a predicted PTLB,including contractile phage tail components and the regulatory/lysisgenes required to regulate production and release of the PTLB particles.

Mass spectrometry data of the Listeria innocua 33090 lysate preparationgave similar results, with several peptides corresponding to a nearlyidentical gene cluster. L. monocytogenes strain 35152, which makesnatural monocin 35152, was chosen as a source for nucleic acid encodinga scaffold for engineering novel monocins and novel expression cassettesfor monocins.

Example 2 Cloning and Expression of the Monocin 35152 in BacillusSubtilis

This example illustrates the cloning of the genes for and expression ofa monocin in a non-pathogenic producer cell.

The monocin gene cluster, from ORF 125 to ORF 142 (SEQ ID NOs: 2-19,respectively), was PCR-amplified from genomic DNA isolated from Listeriamonocytogenes strain 35152 using primers oGL-054 and oGL-057 (Table 2).The PCR product and the vector DG630 (Gebhart et al., 2012) were bothdigested with restriction enzymes AscI and NotI and ligated togetherusing T4 DNA ligase. This placed the monocin cluster between twoflanking amyE sequences which allowed homologous recombination of thecluster into the amyE gene of B. subtilis. This plasmid construct wasnamed pGL-031 (Table 3).

TABLE 2 Primers and oligonucleotides used in this invention. Primer NamePrimer sequence (5′→3′) Primer Description oGL-054GAggcgcgccTTATCTGGTTAATAAGCCGTTTCCGG Forward primer to amplify monocin(SEQ ID NO: 39) 35152 (ORF 125), introduces AscI restriction site.oGL-057 GAgcggccgcTTATCTTTTTCCTGTATTAACTTCTGReverse primer to amplify monocin (SEQ ID NO: 40)35152 (ORF 142), introduces NotI restriction site. oUC-001GAGCGGCCGCTTACATAATTGTTACTTGGCGAAGAG Reverse primer to amplify monocin(SEQ ID NO: 41) 35152 (ORF 140), introduces NotI restriction site.oGL-084 GAggcgcgccGAATTcGACTCTCTAGCTTGAGGCForward primer to amplify Phyper- (SEQ ID NO: 42)spank promoter and lac!, introduces 5′ Ascl site. oGL-085GAgcggccgcTCACTGCCCGCTTTCCAGTCG Reverse primer to amplify Phyper-(SEQ ID NO: 43) spank promoter and lac!, introduces 5′ Notl site.oGL-083 GAgcggccgcTTACATTACAGTTAGCTGGCGTAATGCReverse primer to amplify 33090 (SEQ ID NO: 44)ORF 174, introduces 3′ NotI site. oGL-075CTCTGACATTTTTACAATTTTAGTCATTCTATAACCTCCT Reverse overlap primer forTAATAGTTTCC stitching 35152 ORF 139 with (SEQ ID NO: 45) 33090 ORF 174.oGL-076 GGAAACTATTAAGGAGGTTATAGAATGACTAAAATTGTForward overlap primer for AAAAATGTCAGAG stitching 35152 ORF 139 with(SEQ ID NO: 46) 33090 ORF 174. oGL-086gtgagcggataacaattaGGAAGTGGGAATGGATGG Forward primer to amplify 35152(SEQ ID NO: 47) monocin (ORF 128) for Gibsoncloning into HindIII site of pGL-034. oGL-087ggctagctgtcgactaTTACATAATTGTTACTTGGCG Reverse primer to amplify 35152(SEQ ID NO: 48) monocin (ORF 140) for Gibsoncloning into HindIII site of pGL-034. oGL-089ggctagctgtcgactTTACATTACAGTTAGCTGGCGTAATGCReverse primer to amplify 33090 (SEQ ID NO: 49)ORF 174 for Gibson cloning into HindIII site of pGL-034. oGL-112cgcagtattttcttttgtattccaatttGTtTTcTCTTCcTC Reverse overlap primer forTGaAACcg stitching 35152 ORF 140 with A118 (SEQ ID NO: 50)phage tail fiber. oGL-120 GTTtCAGAgGAAGAgAAaACaaattggaatacaaaagaaaataForward overlap primer for ctgcggg stitching 35152 ORF 140 with A118(SEQ ID NO: 51) phage tail fiber. oGL-103ggctagctgtcgactattatttatcatcctctccatattttttgcReverse primer to amplify A118 (SEQ ID NO: 52)phage tail fiber gene for Gibson cloning into HindIII site of pGL-034.

TABLE 3 Plasmid constructs created in this invention. Plasmid PlasmidName Plasmid Description Backbone pGL-031 monocin 35152 (ORFs 125-142)cloned into DG630 DG630 using Ascl and Notl sites. pUC-001 monocin 35152(ORFs 125-140) cloned into DG630 DG630 using Ascl and Notl sites.pGL-033 monocin 35152 (ORFs 125-139), L. innocua DG630 174 cloned intoAscl and Notl sites of pDG630 (restriction sites restored) pGL-034Bacillus Phyper-spank promoter and lacl DG630 cloned into DG630 usingAscl and Notl sites. pGL-036 monocin 35152 (ORFs 128-140) cloned intopGL-034 pGL-034 using the HindIII site. pGL-038 monocin 35152 (ORFs128-139), L. innocua pGL-034 174 cloned into HindIII site of pGL-034.pGL-045 monocin 35152 (ORFs 130-140) fused with pGL-034 A118 phage tailfiber gene, cloned into pGL-034 using the HindIII site.

TABLE 4 Recombinant bacterial strains generated in this invention.Strain Parent Name Strain Description Strain sGL-064 B. subtilis/monocin35152 (ORF 125-142). BDG9 sUC-001 B. subtilis/monocin 35152 (ORF125-140). BDG9 sGL-068 B. subtilis/monocin 35152 (ORF 125-139), BDG933090 (ORF 174). sGL-071 B. subtilis/Phyper-spank - monocin 35152 BDG9(ORF 128-140). sGL-075 B. subtilis/Phyper-spank - monocin 35152 BDG9(ORF 128-139), 33090 (ORF 174). sGL-092 B. subtilis/Phyper-spank -monocin 35152 BDG9 (ORF 128-140), A118 phage tail fiber gene. 4downstream 118 genes sGL-153 B. subtilis/Phyper-spank - monocin 35152BDG9 (ORF 128-140), A118 phage tail fiber gene. 3 downstream 118 genes.sGL-154 B. subtilis/Phyper-spank - monocin 35152 BDG9 (ORF 128-140),A118 phage tail fiber gene. 2 downstream 118 genes. sGL155 B.subtilis/Phyper-spank - monocin 35152 BDG9 (ORF 128-140), A118 phagetail fiber gene. 1 downstream 118 gene.

pGL-031 was linearized by digestion with restriction enzyme SacII andtransformed into the Bacillus subtilis strain, BDG9 (Gebhart et al.,2012). The transformation protocol was as follows: strain BDG9 was grownin MC medium (Gebhart et al., 2012) supplemented with final 3 mM MgSO₄for four hours at 37° C. The linearized pGL-031 DNA was mixed with 200uL of the BDG9 cells culture and allowed to incubate for an additional 2hours at 37° C. The transformation reactions were plated on LB platessupplemented with 5 μg/mL chloramphenicol and incubated overnight at 37°C. Chloramphenicol resistant colonies were selected and tested formonocin production. This monocin producer B. subtilis strain was termedsGL-064 (Table 4).

B. subtilis strain sGL-064 was cultured using the standard conditionsand monocin production was induced with 5 mM hydrogen peroxide when theOD₆₀₀ reached 0.2-0.4. The protein was harvested as described, andmonocin bactericidal activity was assessed by spot assay. A spot assayis performed by adding 100 μl of target strain culture to 5 ml of TSBsoft agar (0.5% agar), pouring the mixture onto a TSB agar plate, andallowing the soft agar to set. Five-fold serial dilutions of the proteinpreparation are made in TN50 buffer (10 mM TrisCl pH 7.5, 50 mM NaCl)and 3 μl of each dilution, including a sample of the undiluted proteinpreparation, are spotted onto the plate and allowed to dry. The platesare incubated overnight at 30° C. Killing is noted as zones of clearingon the bacterial lawn. Spot assays showed that the monocins produced byand purified from B. subtilis strains sGL-064, expressing 35152 ORF125-ORF 142 (SEQ ID NOs: 2-19) had killing activity on L. monocytogenesstrain 4b 23074.

Example 3 Deletion of the Lysis Genes from the Monocin Gene Cluster

This example illustrates the generation and expression of a constructcontaining a monocin gene cluster but lacking the genes responsible forlysis.

To remove the putative holin and lysin genes (ORFs 141-142, SEQ ID NOs:18-19) from the monocin gene cluster, ORF 125 to ORF 140, SEQ ID NOs:2-17, were PCR-amplified from L. monocytogenes 35152 genomic DNA usingprimers oGL-054 and oUC-001. The PCR product and the vector DG630 wereboth digested with restriction enzymes AscI and NotI and ligatedtogether using T4 DNA ligase. This construct was named pUC-001. Afterintegration into BDG9 as above, the resulting integrant strain wastermed sUC-001. Spot assays showed that the monocins produced by andpurified from B. subtilis strain sUC-001, expressing 35152 ORF 125-ORF140 (SEQ ID NOs: 2-17), after induction as in Example 2 had bactericidalactivity, as evidenced by the presence of spots on a lawn of the target,L. monocytogenes strain 4b 23074. Thus, by removing ORFs 141 and 142(SEQ ID NOs: 18-19), the holin and lysin genes, a larger proportion ofmonocin remained in the cell pellet fraction rather than in thesupernatant of the culture as compared to monocin production from B.subtilis producer strain sGL-064 (Table 5).

TABLE 5 Spot assay of monocin produced in holin/lysin+ vs holin/lysin−recombinant strains. # serial diluted B. subtilis producer Spots: #serial diluted Strain Cell Pellet Spots: Supernatant sGL-064 1 3 sUC-0014 1

Example 4 Changing the RBP (Seq Id No: 17) of Monocin 35152 to that ofMonocin 33090 (Seq Id No: 27)

This example illustrated that changing (“RBP switching”) the natural RBP(ORF 0140, SEQ ID NO: 17) of monocin 35152 to that of monocin 33090 (SEQID NO: 27), an RBP heterologous to monocin 35152, changed thebactericidal spectrum of monocin 35152 to that of monocin 33090, now anon-natural monocin called monocin 35152-33090 (see FIG. 1C).

Based on both the position of ORF 140 (the last open reading frame ofthe structural genes and immediately preceding the lysis genes) and itssequence similarity to that of listeriophage tail fibers, it wasspeculated that this could be the RBP that determines the bactericidalspectrum of the monocin. To determine this, ORF 140 of 35152 (SEQ ID NO:17) was replaced with that the equivalent ORF140 of Listeria innocua33090 (SEQ ID NO: 27).

The L. monocytogenes 35152 gene cluster encoding from ORF 125 to ORF 139(SEQ ID NOs: 2-16) was PCR-amplified using primers oGL-054 and oGL-075.The RBP gene from L. innocua strain 33090, ORF 174 (SEQ ID NO: 27), wasPCR-amplified from genomic DNA using primers oGL-076 and oGL-083. Thesetwo PCR products were then used as template in an overlap PCR reactionto fuse ORF 125-ORF 139 (SEQ ID NOs: 2-16) from L. monocytogenes withORF 174 (SEQ ID NO: 27) from L. innocua. For the overlap PCR, primersoGL-054 and oGL-077 were used. This PCR product was digested with AscIand NotI and ligated into vector DG630 which had also been digested withthe same restriction enzymes. This construct was named pGL-033 (Table3).

Integrants were made from BDG9 as above, resulting in strain sGL-068. Aspot assay showed that monocin purified from the B. subtilis monocinproducer strain sGL-068, expressing 35152 ORF 125-ORF 139 with 33090 ORF174, has a different spectrum than the wild-type monocin 35152 producedby B. subtilis strain sGL-064, validating that changing the RBP genealtered the bactericidal spectrum of the monocin. The monocin with aheterologous RBP, a native ORF 174 from L. innocua 33090, instead of thenatural RBP, ORF 140 from L. monocytogenes 35152, expressed with the35152 monocin scaffold is termed monocin 35152-33090. Monocin35152-33090 killed L. monocytogenes strain 19111, (FIG. 3) which is atarget for natural monocin 33090 and not killed by the natural monocin35152. Thus changing just one ORF (ORF 174, encoding a native RBP) wassufficient to change the bactericidal spectrum of monocin 35152.

Example 5 Expression of Monocins from an Inducible Promoter

This example illustrated the generation of a polynucleotide containing amonocin gene cluster and operably linked to a heterologous induciblepromoter, that is a promoter not found naturally in association with amonocin gene cluster.

To generate a version of DG630 with an inducible promoter regulating theexpression of the monocin genes, the B. subtilis Phyper-spank(IPTG-inducible derivative of spac system SEQ ID NO: 28)(openwetware.org/images/a/al/Phs.doc) along with the gene lacI wasPCR-amplified from plasmid DG481 (Gebhart et al., 2012) using primersoGL-084 and oGL-085. The PCR product was digested with AscI and NotI andligated into vector DG630 which had also been digested with the samerestriction enzymes. This construct was named pGL-034. The monocin genecluster, from ORF 128 to ORF 140 (SEQ ID NOs: 5-17), was PCR-amplifiedusing primers oGL-086 and oGL-087. The PCR product was then cloned intoa HindIII-digested pGL-034 using Gibson assembly (New England Biolabs).The manufacturer's standard protocol was used. This construct was namedpGL-036. After integrating into BDG9 the resulting B. subtilis strainwas termed sGL-071.

Monocin was produced from sGL-071 upon addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) to the culture. Starter cultures ofmonocin producer cells were grown in 5 ml TSB media with 5 μg/mlchloramphenicol in a 15 ml culture tube at 28° C. with 250 RPM shakingand allowed to grow overnight (14-20 hours). This was then diluted 1/200in 200 ml of TSB, 5 μg/ml chloramphenicol, at 28° C., with 250 RPMshaking for good aeration. When the OD₆₀₀ reached 0.2, IPTG was added toa final concentration of 50 μM to induce monocin production. Incubationcontinued for an additional 14-20 hours. Cells were recovered bycentrifugation at 6000×g for 20 min. The culture supernatant and thecells were both saved and processed since there was some “leakage” ofmonocins into the supernatant.

The culture supernatant was processed by ultracentrifugation at 90,000×gfor 3 hours. These ultracentrifuged pellets were resuspended in 1 ml ofTN50 buffer. The cells were resuspended in 10 ml of TN50 with 1 mg/mllysozyme and 250 units of benzonase and then sonicated using a BioLogicsInc. model 300 V/T homogenizer with a microtip. Three 30 s pulses athalf power was sufficient to release PTLB particles. The homogenizedmaterial was then centrifuged at 23,000×g to remove debris. Monocinswere recovered from the supernatant by ultracentrifugation as describedabove for the culture supernatants. In an experiment in which no IPTGwas added to the culture, no monocin activity was observed.

A construct was also made to drive the recombinant monocin 35152-33090from this same heterologous, inducible promoter. The entire L.monocytogenes 35152 ORF 128-ORF 139 (SEQ ID NOs: 5-16) with theheterologous RBP from L. innocua 33090 ORF 174 (SEQ ID NO: 28) wasPCR-amplified from plasmid pGL-033 using primers oGL-086 and oGL-089.The PCR product was then cloned into a HindIII-digested pGL-034 usingGibson assembly (per the instructions of the kit manufacturer, NewEngland Biolabs). This construct was named pGL-038. The resulting BDG9integrant was termed sGL-075. FIG. 2 shows spot assay data of monocinsproduced sGL-075 upon induction with IPTG.

Example 6 Generation of a Non-Natural Monocin Using the RBD of a TailFiber of Listeriophage A118

This example illustrates the generation of a non-natural monocin havingan altered bactericidal spectrum as the result of using an RBD from aphage to create a heterologous RBP.

As provided herein, it was found that it was possible to alter thebactericidal spectrum of a PTLB by making fusions with a portion of anatural RBP and a portion of an RBP of bacteriophage. The N-terminus ofRBP protein was required for attachment of an RBP to the cognatebaseplate of the monocin scaffold, while the C-terminal portion of theRBP, that is the RBD, interacted with a receptor on the target cellsurface. Polynucleotide constructs were designed to fuse the portion ofORF 140 (SEQ ID NO: 17) encoding amino acid positions 1-40, for example,with the portion of the tail fiber ORF 2345 (SEQ ID NO: 21) oflisteriophage A118 encoding amino acid positions 210-357. Four shortORFs (2344, 2343, 2342, and 2341, respectively SEQ ID NOs: 22-25)located immediately distal to the A118 tail fiber gene were alsoincluded.

The monocin 35152 gene cluster from ORF 128 through to the 5′ portion ofORF 140 was PCR-amplified using primers oGL-086 and oGL-112. The A118phage tail fiber gene (SEQ ID NO: 21) was PCR-amplified from phagegenomic DNA using primers oGL-120 and oGL-103. These two PCR productswere cloned in a three-piece assembly with HindIII-digested pGL-034using Gibson assembly. This construct was named pGL-045 and wascomprised of a polynucleotide encoding amino acids 1-40 (BPAR) of SEQ IDNO: 17 and amino acid positions 210-357 (RBD) of SEQ ID NO: 21 plus SEQID NOs: 22-25. The resulting B. subtilis integrant was termed sGL-092.The entire gene cluster was under transcriptional control of thePhyper-spank promoter. The monocin with its heterologous RBP harvestedfrom this monocin producer strain, sGL-092, had an altered bactericidalspectrum compared to that of the wild-type monocin 35152, demonstratingthat an RBD of a phage tail fiber fused to an amino terminal portion,that is a BPAR, of a natural RBP, generated a monocin with aheterologous RBP and possessed an altered bactericidal spectrumdetermined by the heterologous RBD of the resulting heterologous RBP.

Monocins produced from sGL-092 had a bactericidal spectrum distinct fromnatural monocin 35152. (FIG. 3, monocin 35152-A118), and targetedseveral L. monocytogenes 1/2a strains not susceptible to either monocin35152 or any other tested natural monocin.

Example 7 Increasing the Level of Expression of a Monocin by a MonocinProducer Cell

This example discloses a means to increase the level of expression of amonocin by a monocin producer cell.

To improve the yield of the monocin 35152-A118, new monocin B. subtilisproducer strains were generated in which the four short ORFs 2344, 2343,2342, 2341 (SEQ ID NOs: 22-25) that were located just downstream of theA118 tail fiber gene of monocin 35152-A118 were removed one-by-one fromthe monocin 35152-A118 construct. It was speculated that one or more ofthese ORFs could affect the production of monocins. B. subtilis strainsGL-153 included only three of the downstream ORFs, 2344, 2343, and2342, respectively SEQ ID NOs: 22-24. B. subtilis strain sGL-154included only two of the downstream ORFs (2344 and 2343, SEQ ID NOs:22-23). B. subtilis strain sGL-155 included only the first downstreamORF, 2344, SEQ ID NO: 22. These monocins with heterologous RBPsharvested from these B. subtilis monocin producer strains were spottedon a lawn of target strain L. monocytogenes 19111. The data showed thatremoval of downstream ORFs 2341 and 2342 (SEQ ID NOs: 24-25), butinclusion of ORFs 2343 and 2344 (SEQ ID NOs: 22-23) in the monocin35152-A118 gene cluster greatly improved the activity yield.

Example 8 The Demonstration that Monocins are Bactericidal at ColdTemperatures

This example demonstrates that monocins are bactericidal under coldtemperatures.

To determine whether monocins can kill their target strains in the cold,a spot assay was conducted using monocin 35152 isolated from monocinproducer B. subtilis strain sGL-071 and monocin 35152-A118 isolated frommonocin producer B. subtilis strain sGL-154. Once a lawn of anappropriate target L. monocytogenes strain for each monocin was poured,the plates were chilled to 3-4° C. Monocin dilutions were made and alsochilled to 3-4° C. prior to spotting. The chilled monocin dilutions werespotted onto the chilled agar plates. The plates were then incubated for3 days at 3-4° C. The spot assays show that both monocin 35152 andmonocin 35152-A118 can kill their respective target strains in the cold(FIG. 4).

Example 9 The Product of A118 Gene 2344 is a Tail Fiber Assembly ProteinRequired for Optimal Production of Monocin 35152-A118

Many bacteriophage or PTLB RBPs require an accessory protein orchaperone for proper assembly of the tail fiber in order to get optimalactive bacteriocin. This example demonstrates that monocin 35152-A118requires the phage A118 gene 2344 product for this purpose.

Just downstream of the gene encoding tail fiber RBP of bacteriophageA118 are three small open reading frames, ORFs 2344, 2343, and 2342,followed by the genes encoding holin (SEQ ID NO.:025) and lysin. Todetermine whether any of these ORFs encoded necessary tail fiberassembly proteins, four 35152-A118 monocin expression constructs weregenerated, using the same methodology as described in example 6, as setforth below.

Briefly, the 35152 monocin gene cluster, ORF 0128 through to the 5′portion of ORF 0140, was PCR-amplified using primers oGL-086 andoGL-112. The A118 phage tail fiber gene was PCR-amplified from phagegenomic DNA using forward primer oGL-120 and reverse primer oGL-162,oGL-163, oGL-164 or oGL-165 to include three, two, one, or no downstreamchaperone(s). The monocin PCR product and each of the A118 PCR productswere cloned in a three piece Gibson assembly into HindIII-digestedpGL-034. These constructs were named pGL-075, pGL-076, pGL-077, andpGL-078. The plasmids were integrated into strain A8. The resultingintegrants were named sGL-364, sGL-158, sGL-365, and sGL-366,respectively.

One construct included all three putative assembly proteins encoded byORFs 2344, 2343, and 2342; SEQ ID NOs: 024, 023, 022, respectively),another construct included just 2344 and 2343, another constructincluded just 2344, and a final construct had none. These were eachseparately expressed in Bacillus subtilis and the resulting monocinswere assayed for activity on strain 19111. The construct that had noputative tail fiber assembly proteins gave no active monocin particles.The construct that included expression of ORF 2344 produced robustactivity. The construct that included 2344 and 2343 gave monocin yieldsand activity nearly identical to the construct that had just 2344. Theconstruct that included all three putative tail fiber assembly proteinsactually yielded slightly less monocin activity. See FIG. 5. From thisdata, it was concluded that the gene product of ORF 2344 (SEQ ID NO.:022) is a tail fiber chaperone required for production of monocins andwas the only such tail fiber assembly or accessory protein needed.

Example 10 Improved Bacillus Subtilis Production Strain for MonocinExpression

To further improve monocin yields, a modified B. subtilis productionstrain deleted of prophage genes, sporulation functions, and flagellasynthesis, was constructed. B. subtilis strain Δ6 was used, which had aseries of prophage element deletions including prophage 1, prophage 3,SPβ, PBSX, and Skin (Westers et al). Strain Δ6 was further modified bydeleting flagella production (Δhag) and sporulation (Δspollga) togenerate strain Δ8. The M35152 gene cluster, minus holin/lysin, wastransformed/integrated into Δ8, regulated with P_(hyper-spank) upstreamof ORF 0128, as in sGL-071. The resulting strain sGL-157 (M35152) hadimproved monocin production (typically 5-10 fold) over BDG9-basedcounterpart. See FIG. 6.

The methods used to produce this strain are detailed as follows.Bacillus subtilis knockouts were made following the methods described inTanaka et al. (Tanaka K, Henry C S, Zinner J F, Jolivet E, Cohoon M P,Xia F, Bidnenko V, Ehrlich S D, Stevens R L, Noirot P. 2013. Buildingthe repertoire of dispensable chromosome regions in Bacillus subtilisentails major refinement of cognate large-scale metabolic model. NucleicAcids Res. 41:687-699). Strain Δ6 was described by Westers et al.(Westers H, Dorenbos R, van Dijl J M, Kabel J, Flanagan T, Devine K M,Jude F, Seror S J, Beekman A C, Darmon E, Eschevins C, de Jong A, BronS, Kuipers O P, Albertini A M, Antelmann H, Hecker M, Zamboni N, SauerU, Bruand C, Ehrlich D S, Alonso J C, Salas M, Quax W J. 2003. Genomeengineering reveals large dispensable regions in Bacillus subtilis. MolBiol Evol 20:2076-2090) and is a prophage deletion strain. In order tomanipulate strain Δ6 further, the cat gene, which was a remnant from theoriginal pks operon knockout, was removed. First the upp::kan marker wasamplified from Bacillus subtilis strain TF8A λPr-neo:Δupp with primersoDG1013 and oDG1014. The PCR product was cloned into pETcocol linearizedwith NotI. This plasmid was then linearized with Spe1 and transformedinto Δ6 and selected for kanr. This strain was termed BDG243. To deletethe cat gene, the phleomycin cassette was amplified from pUC18 phleocassette (Tanaka et al.) in a sewing PCR reaction with two flankingregions from Δ6 using the primers oDG1001 and oDG1002, (left flank)oDG999 and oDG1000 (phleomycin cassette), oDG1003 and oDG1004 (rightflank) and the three pieces combined by amplification with the twooutside primers oDG1001 and oDG1004. This PCR product was transformedinto BDG243 and selected on phleomycin plates followed by screening forkanamycin sensitivity. This strain is designated BDG247. The phleomycinmarker was deleted by growing BDG247 in LB without selection for 4hours, plated on kanamycin, and colonies picked and screened forphleomycin sensitivity. This strain, BDG252, was a markerless knock-outstrain, useful for making further modifications. To delete hag, the 5′flanking region of hag was amplified with primers oDG1019 and oDG 1020,the 3′ flank amplified with oDG1021 and oDG1022, and the pleomycincassette amplified with oDG999 and oDG1000. The three PCR products werecombined and a sewing reaction performed with oDG1019 and oDG1022. Thisproduct was transformed into BDG252, selected on phleomycin, and thenscreened for kanamycin sensitivity to create BDG253. The phleomycinmarker was again deleted by growing BDG253 in LB without selection for 4hours, plating on kanamycin, and screening colonies for phleomycinsensitivity to create strain BDG255. To delete spoIIga, the 5′ flank wasamplified with primers oDG1023 and oDG1024, the 3′ flank amplified withoDG1025 and oDG1026, and the phleomycin cassette amplified with oDG999and oDG1000. The three products were combined in a sewing reaction usingprimers oDG1023 and oDG1026. After transformation, selection onphleomycin, and screening for phleomycin resistance, the resultingstrain was named BDG256. The phleomycin marker was deleted, again bygrowing BDG256 in LB without selection for 4 hours, plating onkanamycin, and screening colonies for phleomycin sensitivity to createstrain BDG257, also known as the Δ8 strain.

Example 11 The Spectrum of Monocin 35152 and 35152-Δ118 EncompassImportant Foodborne Serotypes

Most human illness caused by Listeria in North America is caused by twopredominant serotypes, 1/2a and 4b (Nelson, K. E., D. E. Fouts, E. F.Mongodin, J. Ravel, R. T. DeBoy, J. F. Kolonay, D. A. Rasko, S. V.Angiuoli, S. R. Gill, I. T. Paulsen, J. Peterson, O. White, W. C.Nelson, W. Nierman, M. J. Beanan, L. M. Brinkac, S. C. Daugherty, R. J.Dodson, A. S. Durkin, R. Madupu, D. H. Haft, J. Selengut, S. Van Aken,H. Khouri, N. Fedorova, H. Forberger, B. Tran, S. Kathariou, L. D.Wonderling, G. A. Uhlich, D. O. Bayles, J. B. Luchansky, and C. M.Fraser. 2004. Whole genome comparisons of serotype 4b and 1/2a strainsof the food-borne pathogen Listeria monocytogenes reveal new insightsinto the core genome components of this species. Nucleic Acids Res.32:2386-2395. Accordingly, the bactericidal activity of monocins 35152and 35152-Δ118 were tested against a panel of independent Listeriaisolates. See Table 6. Monocin 35152 killed 4b strains, whereas monocin35152-Δ118 killed 1/2a strains. Therefore, a biocontrol agent thatincludes monocins 35152 and 35152-Δ118 may be used to kill thesefoodborne pathogenic strains.

TABLE 6 Bactericidal activity of the monocins on a panel of Listeriastrains. Sensitive Sensitive to to Other M35152 M35152- Straindesignation Source Serotype (recombinant) A118 15313 ATCC 1/2a No Yes35152 ATCC 1/2a No Yes 19111 ATCC 1/2a No Yes DP-L4056 10403s Dan 1/2aNo Yes phage cured Portnoy DP-L3633 EGDe Dan 1/2a No Yes PortnoyDP-L3293 LO28 Dan 1/2c No Yes Portnoy DP-L3817 1993 Dan 1/2a No YesHalifax Portnoy DP-L1171 Dan 1/2b No Yes Portnoy 23074 ATCC 4b Yes NoDP-L185 F2397 Dan 4b Yes No Portnoy DP-L186 ScottA Dan 4b Yes No PortnoyDP-L188 ATCC Dan 3 No No 19113 Portnoy DP-L1173 Dan 4b Yes No PortnoyDP-L1174 Dan 4b Yes No Portnoy DP-L1168 Dan 4b Yes No Portnoy DP-L1169Dan 4b Yes No Portnoy 19119 ATCC Yes No (ivanovii) 33030 ATCC Yes No(innocua)

Example 12 Monocins are High Molecular Weight Bacteriocins withTp901-1-Like Tail Structures

All high molecular weight bacteriocins described to date have beenrelated to either contractile Myoviridae-like structures (R-type) orLambda-like tail Siphoviridae structures (traditional F-type). Monocinswere shown to be F-type bacteriocin based on a lack of a contractilesheath protein and electron microscopy. However, as shown herein,monocins were determined to be closely and specifically related to thetail structure of phage Δ118, a TP901-1-like phage (Cambillau, 2015).Comparison of a monocin major tail protein (SEQ ID NO.: 8) to those ofTP901-1-like phages (SEQ ID NOS.: 30, 31) including Δ118 (SEQ ID NO.:32), and comparison of monocin tape measure protein and baseplateproteins (respectively SEQ ID NOs.: 11, 12, 13) to those respectiveproteins of phage Δ118 (SEQ ID NOs.: 33, 34 and 35), indicated thatmonocins as described herein were structurally TP901-1-like. Inaddition, three monocin regulatory proteins (SEQ ID NOs.: 3, 5 and 6)were shown to have Δ118 homologues (SEQ ID NOs.: 36, 37, 38). Acomparison of protein sequences encoded by the monocin gene cluster tothose encoded by the Δ118 genome is shown in FIG. 7. Thus, monocins fellinto a unique class of TP901-1-like high molecular weight bacteriocins.

TP901-1-like phages have a distinct baseplate structure wherein thereceptor binding protein (RBP), a homotrimeric protein, is arranged insix groups with three “tripods” each (see Bebeacua C, Tremblay D, FarencC, Chapot-Chartier M P, Sadovskaya I, van Heel M, Veesler D, Molineau S,Cambillau C. 2013. Structure, adsorption to host, and infectionmechanism of virulent lactococcal phage p2. J Virol 87:12302-12312;Collins B, Bebeacua C, Mahony J, Blangy S, Douillard F P, Veesler D,Cambillau C, van Sinderen D. 2013, Structure and functional analysis ofthe host recognition device of lactococcal phage tuc2009. J Virol87:8429-8440; and Cambillau C, 2015, Bacteriophage module reshufflingresults in adaptive host range as exemplified by the baseplate model oflisterial phage A118. Virology 484: 86-92).

This results in a total of 54 RBPs per phage particle (3×3×6). R- andF-type bacteriocins are known to possess just six copies of singlehomotrimers (18 total copies). This is the first example of aTP901-1-related structure capable of functioning as a high molecularweight bacteriocin.

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

Listeria monocytogenes strain 1/2a 35152Motiocin gene cLUster (wild type) SEQ ID NO: 001TTATCTGGTTAATAAGCCGTTTCCGGTTTGAAAACTATCTACTTGATCGAGTAGATTCTCGTGCGAGCTAATGGTGATGGGCTTTTTGGAAAGCAGTAGTCCGCTTTCCCGTAATTCTTCTAACACTTCAGTTACTCTAGCTCGACTCGAACGACAATAATCAGCTAAGAACGTTTGCGTGAcATAGACTGGAAGCTCAATTGAGCCTTCTGTTTGATGCAAATGAAGTAGCTCAATAATTICCACCAAAATACGCGTTACTTTGACTATGGGTGCTTGATTAATATTCTTGTAATTTTTTGATGTAAATAAATATCTAACAATAACTTCATCAAGTATAAGATGGAAAAATTGTGGATTTGCATATAAATAATTGAGAAGAAACTCGCGGTCAATAAATAGAACGGTACCGTTTTCTAATGCGCGAACACGGTATTCAGTAAGTTCgGGCGCTGTTTTTGTTTCGAGTAAACTTTCCATTCCAAAAAGCGCGTGTTGACCAATCAACGCATTTATCATCCAGCGGCCTTCATTTGTAGGACCTTCTAAAACAGCCtTTCCCTCTAAAATAGCACCTATTTGCGGCGCCTTTTCTTCGTAATCTCTATATGAATGAATAATTTCTCCTTTTTTAAAAGAAATTTTACTTGAATAATCACTAGAAAAAATATCTGAAAAATCTATCGTGTGAAGCATTATCAAAACTCCTATACTAAGGTAAAATTGGTATTTACCATATTACTGTACTGCTATTTGTGtCAAAAAGATACCGAATAAAAATCGTGTTCACAATTTAGACATGAAAAAGTCACTTTTTAGCAACAATTTTAGTGATTTTATCAATAAAAACTTACTTTTTCTTCCCTGGAGAGATATCCAACCAAAAAAAGACCCTCCgTAGATAGGAGAGTCTTTCAAAACTTCTTGTAAACATCAATGGAATTGCCAAAATTAATGAAATATCCACCATAATCATACATTGGACCATATTTTTCCGCGTAATGAGAAACTGTATGGCGCAAAAATTCTTCTGTTACCTCTAAAAACTCCGCTACTTCAAAATATTCTCGTAAGCCTAGATAAAAACAAGCAATGATGTCATCAAGcGGTATTAGTTCCTCATAACCTTTGCGACGAGCGAAAATTTCTTGTTTTTTATCCATGATTGTTTCCTGTTTGGTGATATTACCTACAGTGTATTTGTAATGCATCAATTCTTCTACTAAAACACAGCCTTTTTCAATGGAAGATTGATCCTTACTAATAAAAATCATCCCATTTAAATAAAGTCCCGGAAGTTTGTAAGGCATTTTCTCTTCTCTGATTGTTACCTCATCTTGGTATTTATTTACCAATTTTTCGTACAATAAAATCACATCCCGCGATGGAGTTTTTTCTTCATCTCTATATAAGCTAAAATATCCCGCATTTCTTCCTCGGTTACGTCATCATCAATATGGGCTGCAATTGTTACCAAGCGTTCATCAACAGGGGGTAAATTTCTTTCAGGAAAAAAGTCATCTAATCGTTTGTTGAAGATTTTTGCAAGTTCAAATAAAACATCTTGATTTGCTTTTCGATCACCATTTTCATAACGGCTAATAGTTTGGCGAGTTGTATGAAGCTTTTCTGCTAATGCTTCTTGATTTAAGCCTCGTTCCTCGCGATATTGTTTTATTTTATTTCCTACAAATTTATTTAGCTCCATAATAATTGTCCTCCATATGTTTATGGCTTCAGTATAGCACTTTTAGCACCAAAACGGAACTTTTTCGAACATATTTGTCGGATAAAGTTTACCAGTAACCAAAATGGTGCTATAATATTTGTAGAGCTAGAAAATAAAtGCGGCTGATTTTGCACcGATAGCACCGAATcGGTGACAAaaCTAcTATATTAATTTATGAGATGGAAGTGGGAATGGATGGATAGAAAACTTTTAAAAGAAAAGCAAATCCAACTAATTTTTCAACTAGAACAAGAAGAAAATCGATTTATTCGAAAGCGTTTGATAGAAGAGTTGGAGTTTTTTGAAGCACTTGGGGATAGGGAAAAAGGACTTCTAACGGCGGAGCAAAAGTTGCTTATTTTAaCACCTAGTGAGTACCGAGAATACAAAAGAACCAAATCAGATGTGCAAATTAGTAGGATAATTGGAGTATCGAGATCGTCACTTGCAGAATGGAAaCGAAAAAAAGGTTTAAATAGAAAAAAGTCGCAACCGGTTCAGCAGGAAATGATTGATGTATTAGCTTTTCATTTAGATAAAACAAAAGAAGAAATTGGCGCTTTACCTGCTTCGGCGATTGAATGTCAGTATGAGGCTTTTGTGATTAATGAAGCCAATAATTAAGGAGTGATAAAATGCAGGTTTTAGTTTTACCAGAAAATAAGGATATCAATTATATAAAAACGGTCCAtGAAGTAAAACGATTTTTTGCGGATTTCGAGAGATTTCGGATGATTACGGGGTTATCAAAAAAGCCACATTTACTTAGAAATGGTTTTCTGGAAGAGCCGCAGTTTGAGCCGGTAGCATTTTCTGCTAGACATAATAAAGAgGTCATTTTGGAAGCGCGATGGTTGGTAGAGAAATATACTGAAATGTTGAATCAGATGGATGATTTATATCGAAGATGGTTGGTAGAGAAATATACTGAAATGTTGAATCAGATGGATGATTTATATCGAACTATTTTGATGGAATGTTACGTGGAACGAAAACAAGATGTGGCGGTAATGATGGATTTACCGTATGAAATTGCCCAGTTTAAACGGATAAAAAAACGgGCAGTGCTAGAACTTGCAACGCTAATGGGAATTTTAGTAAGGAAATGATGATACTTTCGTGATATTTTGAAACATCATTTTCCTATTAATATAGAAGTAAGCTAATTGTCCAGTAAGCGGATGACAATAAAAGCTGCATCAGAATGAAGGTGCACCGATTTTCTGATAATACATGATGTTTTACAAGAAATTTGTTTTTATGATTGGATTTAAATCCGTTGAGATAAACAAATATTCTATTTTGGAAAGTAAAGTTCGGAGGAATAAATTATTAAATGTGGTCTTGACCGAACTTTGCTTTCTGTTTTAAAGGAGTGAACGCTTGGTGAAGAGTTTGAGCTTCATGAGAGTTTTGGAAG CAGTGAGAAcAATGCTCCAGGAAAAAGGCGGACTAGATGTTTCTATTGTAATGCGTaACCAAGTGGAAATGCCTACAACGATGATCGAGATGATTGATCAAGAGGAAGAAGAAAGCCAAACTGCCTGGAAAGAAAAATACCGTTTTGCtATaCATCATTATACAAATGAAcaGGACtTAGCGGGgGTCGAgAAGATtGATACGCTTATCCAAAtgGGtTTCATtTTTGCCcGAgGGATACAAATTAgTCGCaGTTCGACATTgtGGAAAACAAAATTTAGTCAAAGAAAATACGTTAATTCACGCAAAAACCAGTTTTGAAGTAAGTATTTGTCGTGAgTTAAAAGTAAAAATTTAGGGGGAAATATTAATGGCATTTGAAGAGAATTTATATTGTGATTATACACCGGGAGCTGCTAAAGCGGTCGCGGGGAAAGATGTAATTTTAGCAGTTTTTAACGCAGCGGGGGAtAAACTATTgGCtGTTGCGGGCCAACAAGGTCTgACTGTAAACCGTTCTAAAGATAGtATTGAAATTACATCcAAAGATACAGTgGGCGGATGGAAATCCAAAATTGGCGGTATGAAAGAATGGTCAATTGAAAATGACGGgTTATATGTCGCTGATGCAGAGTCTCACAAAGAATTGGCGAAATATTTCGAAAGTGATAGCCCAGTTTGcGTGAAAATCATTAATCAAGCATCTAAAAAAGGTCTTTTCGGTGGTTTGGCAATTGTAGCTGACTATAGTTTTGAAGCgCCTTTTGATGAAGCGATGACTTACTCTGTAAAACTAGACGGAATGGGCGCGCTTGTTGATTTAACGATTACTGAGGGCGGCGACCAAATGCCCGGCGAAACACCTGTAGCACCAGCAGAATAAAATAGAAAGCCACTGAAATAAGTGGCTTTCtCTTAGGAGGAAAATAAATGTTTGAAGTGAATGATACAACTTATATTTTACGATTTAATAAACAAAAAGTTAAAACGGTGGAATTAACATCAGGGATTAGTTTAGTTGCAGCTTTGACTGCGAATAAAGGGATTTTGAGCTATCAAGTGATTGAAACgCTATTTGTTTCAGGACTTGTGGAAGAAAAAGGCTTAGTACCTGTAAAACAAAAAGAAGCcTTGGAGATTTTCGATAAATTAGTAGAAGAACAAGGCTTAATTTCcCTTAATGTAGCTGTTATTGAGAAATTGCAAGAGGATATGGGTTTTTTGTTCCGTTAAAACAGATTGAaTTTGAGTATTTTGGTGCTGAGGACGAAGAAGTGGATAGTGAAATGAACCATGATTTtGATTTGGAAAAACAgTTcGCTTTTTTTGTAGTcAATTTTCAaATGTCCAAGCATGATTTTGAAGAACTTACTGAAGTGGAGAAAAATTTCATCATGAAAGAATGGGAAAACAAGGTGATTTTTGAATCTACTATGCTTCGAAATGCAGTTTTAAATGCGGAACAAAATCTCAATCGAAAACGAAATTCaCGTTTTATCGACTTGCATAAAAAACGTCAGAAGAAAGCCGATGTTAATTATACAGTAAATGCACTTCAAGCAATTTCCGATAATGAAGCGAAGGAAGGTAAAGCGTGGATTGATCGGATTTATGGTGCAAATGGGTTGCGAAGACCTAAAAaTAAAGAAGAAAGGGGGAAAATGAATGGCGGAgTCTAAAAGTaTTACATTTGAACTgAACGAGAGCGTTTTAACAGCGCAAGTTGGCAGGCTAGATGAgATGGCGATGGTTGTAGAGCggCGgTTTTCAGAGCTCAAAATGACTATTGAAGATGTTGGAAATGCTGATCCAGGTTCGAAAATTTCCGAaTCTTTAGGTGGGCTGCAGTCTGGGCTTGGCACGATTAGTTCGGCGTTTGGACAGCTGGGTTCTAGTAGTGAGGCGATTACATCcGGATTCGGTACTGCGGTTGGTTCTGTTGGTGGAATCACGGATGCGTTTAAAAATCTAGGTTCAAGTGTGCAAAATGGTACGTTATTTTCAAGCTTGGCgACtGGAATTGGTGGCATGAGTACgATGCTTGGTGGAGTATCTGGCGGcGTTCAAGGAATTACAAATCTAGCTAGTGGATTTATGGAATTGAAGAATCATTTAGGCGGTTTGATGTCTTCTATTGGCGGCGTTGGTGGAATTATGGGTAAACTGACTTCTCCAATGGGGTTAGTAATTATCGGGATTGTTGCGCTAGTTGCTGCTTTTACGTACTTGATGACGACGAATGAATCGTTCCGAAATACCGTGATGTCAGTCGTAACGCAGGTTGCGCAGTTGTTCGGGCAACTTGTCGCTAGTTTAATGCCGATTATTATGCAAATTGTTACTGCGGTTATGCAAATTGGTGCCGCGTTAATGCCGATgGTTATGCAGTTTATTAGCTTTTTTGCCCAGTTGTTAGCTCAATTAATGCCATTTATTAATATGCTGATTTCTATGCTTATGCCTGTTATTATGCAGATTGTTCAAGTTGTTATGTCGCTTGTTTCAGCGTTATTACCAAGCATTATGACAGTGATCCAAGGCATTATGAGTGTTATTCAATTTTTAATTCCGATAATTATGCAAATCGCGACGGTGGTTGTACAAATTGTTGTAACGATTATTTCTTATATAAGTAAAATTATGCCGATTGTtATGACGATTATTGGCGTTATTGTTTCGATTATCACaACGATTATTAGtTAcGTcGTTATTATTGCgACGACgATTGCtAGtGTTATTGGGAAAATTATTAGCTTTATTGCgAGTGTTATTACgGCGGTTATCGGGATTGTGCAACCAATTATTGCCTTTATTACCAATATCTTTACGACTATCGTGACAATTATTGGTGCAGCTTTCCAAATGGTATTTACTGTTGCATCCAAAATTTGGAATTCCATTATGTCGACTATTTCCGGAATTATTGAcGGAATCAAAGCAGTCATCACAGGTATTTCTACTACAGTTTCATCAGTGTTTAAcGGAGTGAAGCGCATTATTACAGGTGTTTTTGACGGAATCAAAAGTGCTTGGGGTGGTTTAACTGATTTTGTgGGAAATATTTTCGATGGTGTTTCAAgTGCAATTCAAACAGTGGTAGACAATGTCAAAGGTTTTGTAAACGTgGTAATTCGAGGgATTAATGGAGCCATTGGTTTAATTAATAAGATTCCAGGAGTTGAAATCGGCAAAATACCGCAATTAATTTCCGGAACAACAAATTTCCAAGGTGGCTTTGCTCGAATGAATGAAGGCGGCCGAGGTGAAATGGTTGTTTTACCGTCTGGTTCTCAAGTAATTCCGCACGATGCAACGATGAAATACGCAAGAGAAAGTGCGCGCGGAAATAAATCAATGCTITACACGAGTCAAGGCGCTGATTTGGCTAGAGTTGAAAATCTTCTCGAGCGCTTACTACAAAAAAATCCTGTAATCAAAATGGATGACAAAGTGGTAGCTGAGGTAGTTAGCCGTAATCAAGCTAACTCATTTGATCAGTACAACTATACAATGGGAGGTGCAGCTTATTCATGAGtGACTTGTTTTTAGAATTAAATGGAAAAGTGCATTCGCTTAGTGAGACATTTCCAGGTCTTTCTGTACAAGAAGTTTCGAGACAAAGTCCCCAGTTAAGCATGGAAACTGCTGAAATAGCTGGGACTGATGGGGTTATCCCgGGAATGACCCAATTTAAACCGTTTATCTTTTCAGCAAAATGTAATTTGCAAGCACTTGATATTCCaGATTAtCATTTGGCAGTCAGAGAAATTTATGAATTTTTATTTCAACGGGATAGTTATTATATTTGGAGCGATCAAATGCCAGGAATTCGGTATGAGGTGCATCCTAAACCAGTTGATTTTAGTCGAGAATCGGATCGTGTTGGtTTACTCACTATAGAATTTGATGTAITTAAAGGCTATGCGGAGTCACGTGGCACGAGCCTTGACCCaATGACTTTTGAAGTGGATTTATGGCAgATGGGAATGAATTTATCGAACCGTGATGATTTATTTTATGTTTTTAGAGAAAATACATTTCGGGTCTATAATGCGGGGAGCGACCGTGTTAATCCACTGATGCGACATGAATTgGATATTGCTATGACGGCGAATGGGACACCAACGATTCATAATCTTACAACGGGAGAATCCTTCGAGTATCGGAAAGAGCTACAAAAAACAGATGTTTTACTGTTaAACAATATTTATCCACTTGTTAATAACCGcCGTGTTGGAAAAGATACCAATCATGGGATTATCACCCTTGAAAAAGGCTGGAACGATTTTGAAATCAAAGGTGTAACGGATGTAACGATTGCTTTTAATTTTCCGTTCATTTATCGGTAGGTGATAGATATGGATTATGTGATTATTCAAAGTATGGACAAAGAAGTGGAAGAGATTCTaACAGACATtGATTACGGCTCCTTTTCCTACGATTATGAAAAAAATACAAGTCGTGCTATtTCGTTTACTGTGAAcAAAACGAAACAGAATGCAGCAATTTTTGACTTGGTAGGAAATGAAGCAATTTTAACATATCAAGGGCAGCAATTTGTTATTAAAAAATGTACGCCAAAATCTATTGGAGGAACAaTTTCAAAGCAGATTACGGCCCAGCATATTTGTTATACAGTGCAAGATCATGTGCAGTATAACGTGAAATCTGGACGAAAAAAATATTCGATTCAAACGGTATTGGAATTTGCGTTACAAGATAATGTACTAGGATTTTCTTATGAAATTCAAGGGAGTTTTCCTTTAGTTGAATTAGAGGACTTAGGAAATAAAAATCTGCTTAGAGCTAGTGAATTTATGTTTGGAAGAATTCGGAGCAATTTTATTTGCAGATAATAAAAAGCTTTATTTTTACGATGAAAAAAGtTGGTATGTAAgGACAGAGAAGCAATTTCGTTATTTATATAATACAGAAGAAGTTTCGGTGGATACGAACACAGACAATTTGAAGACGGAGATAAAATGTTACGGCAAGCAAAAAGAGAATGCCGATAAGCTGACTGGAGATAATAAGTACATGGCGGTTGTCACGTATACTTCGCCaAATGAGGCTATTTACGGGAAACGAATGGCAAATGCtAAAAGTGATGACAAAATCACGAACAATGATGACTTATTAATTTTTGCAAAGAAGCAAATTCTAGATGTTCCgGAgACgGCGCTTACTATCGCTTACAAAGGAAAAGAACCTGTTTCAGAGCGGGATGTTTGGTATTTCATTCATGAACCGATGGGGTTTGAAACAGAAGTAAAAGTAACGAAAATTAAATCGAGTCATCCTTGGAGTAAGAAGTTTCAAGAAATTGGCTTCAGTAATTCGCGACGgGATATGGTCCGAATTCAAACGCAAATTGCTAATCAAGTGAAAAAAGCGAGCGTAGATACAAATAAAATtAATTCGTTTTCGAGCATCGCAATGAATGCTTATGATTCACGAATTTTAACGGAAGTAGTAGGTGTGGTAGATGGCGACTGAAATTAGAGTGTTAAAAAATGTAGATGATACAGTTTTCTATCCGAAGACACATGTAACGGCCGTGGAAGGTTTAGACTCGGCTACAACTACTACATCTGGATTAATGCCCGCCAGCGACAAAACGAAATTAAATGGAATCGAAGCTAATGCAGAAAAAAACAATGTGACTGCAATCGATATTGCCAATTGGAATAAAAAAACAGGACGCAATTTTGGTTTCTGAAAATGGTTCTAATTTCAAAATAACTGTCACAAATGCTGGTGAACTAAAGGCAACAAAAGTGGAATAGGAAGGAGGTTGCGTATGAAGTTGGATTTATGGAAATGGGAAATGCTTCTTCAAGGTCGAGAATTTAGAAATAAAACAAATGACAACTCTGCAAAAATTGATGGATTGGTCCGATTTTATTTCAACAGGTTTAAGCGCGATTTATGTCTATGTAAATAAAGCGGATGCTACCTTAAATAACAAAATTGATACCGTGGATAAAGCAGTAAATGCAAGGGTTAATGAGCTGATTAGCGGGACAGAGCAGCTAAGTGAAGTGGTTGATGCGAGATCAGATGCGTTTGGTGCACGATATCCTGTGCTAAGAGAACGTTTAAACCAAGAACAGCTTAACTTTAGCAAAAAGAGCACGATTCAATTTGATGCGAGTACTATCATAAGTATGGAAAAACAAGATATTGGGCTGCTAACAAGTAAAAAAATCTCAGAAGCGCAAACCGTATGTTTTTTAAATATATCAAGCCTCGATGAAGAAGCaGATATTGTtCTTGAAAAAACAGGtGAGACAAGCTTCTCaGAcAATTTAACgAGcCTAGTCTTTGCgAAAATTGGAACGAATGAACGCTACCAAATGGAGCCAGTTGGTGCATAAAGGAGGAGTGAGCgATGACTGAAATAAAGCGAATGCTaCAGACAAAAGAAGATAATTCAAAAGAACAATTTTATCCAGAGACGCATGTTGCgGGGATTGTCGGGTTGACaGAATATGTGTCAGGTCAGCTTCCGACgGGtGTGGTCAGTGTgAATGGTAAGGCaGGtCGCGTGTTaCTgGATGCtGAAGACGTtCACGCTGCaAAAAAaAGCCACAcCCAtGAAGTCGCAACATACACtACGGAtGGCTTTATGAGTTCTTTTGATAAACAAAAGATTGATCAATTAGTTTCACCgGAAGCTGGCGTGACAAGCATAAATGGTAAAACAGGGATTGTTGATTTATTCGCATCGGACTTAGATGCAGCAGAGATAAACCACACgCATGCAGAAGCaACtACTACAGAAAGCGGTTTTTTatCAAtcGAcGACAAAGAAAAAtTAGAtGCGATaCAAgTAATCGCGCTGGAAACTATTAAGGAGGTTATAGAATGACTAAAATTGTAAAAATGTCAGAGAAgAATGAACATGGAACtcTaGAaCAATTCTATCCAGAAACACAtGCaGAGGCTGTTAAAGGaCTTGTGtcgGTTtCAGAgGAAGAgAAaAACaAtTTGGGATCAAAAGGANAGTACGGCGGGTGCAGAGCAAAAAGCAAACACAGCCTTAAATAGTCTCTAAACTATTATGTGGATACGATAGGIGAAGGAACGGTGATTTTTAAAGGCGCTAACCTTATGGGAGCTGGCCAATCATTTAAATGGGACGCTTCTAAACTGAAATTTGGGATGACTTTGTTATTTAGTCGCTATGATGCGGCAAATAATACACCGCAAGATTATTATTATCATTCTGTATTCTTATcTAAGGCACAATTAGTTGAGCTTGCAGGAAAAGGTATTTTAGTTCAAATGCCATCAACGACTTATGGAGATCGAAAATATCTGTATGTTICAACAACTGGGTTATCTGGACATTTTGATAATTCGAATTATGCGGCTTGGGCTCTGCGCCAAGTAACAATTATGTAACTAAAAAGGAGGTTTTCCATGGAAGTCATACTAAAATTCGGGATTTTAGGTTITGGCGCGATATTTGGATACTTGTTTGGGGAAGTGGATTTATTGGTAAAAGTGCTGGTGTGCTTTATTGTAGCTGACTATATTTCTGGGCTACTCGCTTCAGGGTATCTTGGGGAACTTAGCAGCAAAATGGGTTTCAAAGGAATCGCGAAAAAAATCGCTATCTTAATTTTAGTGGCTATTGCGCATCAAATAGATTTGATTCTGGGAACGCATAATACAACCTCGGGATGCGGTTATCTTTTTCTATTTACTCGAATCTAGCTGATTTCTATCTTGGAANATTTCGTTCGAATGGGAATGAAGGTCCCGGAAGTATTGAAAAATTTAATTTTGATTTTCGATGCAAAGTCAGGAGAGGATGAGGAAAAACATGACAAAGATATGGATTGACGCTGGACAcGGTGGTAAGGATTCAGGtGCGAGTGGTAATGGACTTGTTGAAAAAAATTGGGTACTAACTGTAGCAAAACAACTTCAAACAGAGTTAGTTAAAGCCGGTTTTGAAGTGGGAATGACAAGAACAAATGATACATTCTATGAATTAAGTGATCGTGCGAAGAAGGCGAATAGTTTTAAAGCTGATTTATTTATTTCGCTTCATTTTAATGCTGGTGGCGGTAAAGGATATGAAGATTATATTTACACATCCGTCCCGGCTGCAACGGTAGAAATACAGAAAATAATTCATAAAAATATTATTACTAAAGTTACTAAACACGGAATGAATGATCGTGGAATGAAGAAAGCTAATTTCGCTGTATTAAGAGAAACAGCAATGGATGCTATCTTACTTGAAGCTGGATTTTGCGATAGCACTGATGCATTAATTCTTGAGAAGAAAGCTTATCAAACTGATTATTGTTTAGGAATTGTATCAGCAGTACAAGAGaTTTTTGGGGCTATGGTAACAAAATATAGGGCAGGCAAATATTTGACAAGTGACGATGCTATATCAGGCACAAATATTAAAGGATATTTAGAAGCAGGAACAAAGGTTTTTGTTTATAAAGAAACAGAAAAAACGCTTAATTTAACTACTACAAAGGGTGTTCCAGGAAGCTGGGTTTTAAAAACAGAAGTTAATACAGGAAAAAGATAA Listeria monocytogenes strain 1/2a 35152Monocin ORF 125 >transcriptional regulator SEQ ID NO: 002MLHTIDFSDIFSSDYSSKISFKKGEIIHSYRDYEEKAPQIGAILEGKAVLEGPNEGRWMINALIGQHALFGMESLLETKTAPELTEYRVRALENGTVRALENGTVLFIDREFLLNYLYANPQFFHLILDEVIVRYLFTSKNYKNINQAPIVKVTRILVEIIELLHLHQTEGSIELPVYVTQTFLADYCRSSRARVTEVLEELRESGLLLSKKPITISSHENLLDQVDSFQTGNGLLTRListeria monocytogenes strain 1/2a 35152Monocin ORE 126 >hypothetical protein SEQ ID NO: 003LYEKLVNKYQDEVTIREEKMPYKLPGLYLNGMIFISKDQSSIEKGCVLVEELMHYKYTVGNITKQETIMDKKQEIFARRKGYEELIPLDDIIACFYLGLREYFEVAEFLEVTEEFLRHTVSHYAEKYGPMYDYGGYFINFGNSIDVYKKF Listeria monoeytogenes strain 1/2a 35152Monocin ORF 127 SEQ ID NO: 004 >putative repressor proteinMELNKFVGNKIKQYREERGLNQEALAEKLHTTRQTISRYENGDRKANQDVLFELAKIFNKRLDDFFPERNLPPVDERLVTIAAHIDDDVTEEEMRDILAYIEMKKKLHRGMListeria monocytogenes strain 1/2a 35152 Monocinn ORF 128 >antigen DSEQ ID NO: 005MDRKLLKEKQIQLIFQLEQEENRFIRKRLIEELEFFEALGDREKGLLTAEQKLLILTPSEYREYKRTKSDVQISRIIGVSRSSLAEWKRKKGLNRKKSQPVQQEMIDVLAFHLDKTKEEIGALPASAIECQYEAFVINEANN Listeria monocytogenes strain 1/2a 35152Monocin ORF 129 >antigen C, transcriptional regulator SEQ ID NO: 006MQVLVLPENKDINYIKTVHEVKRFFADFERFRMITGLSKKPHLLRNGFLEEPQFEPVAFSARHNKEVILEARWLVEKYTEMLNQMDDLYRTILMECYVERKQDVAVMMDLPYHAQFKRIKKRAVLELATLMGILVRK Listeria monocytogenes strain 1/2a 35152Monocin ORF 130 >antigen B SEQ ID NO: 007LSFMRVLEAVRTMLQEKGGLDVSIVMRNQVEMPTTMIEMIDQEEEESQTAWKEKYRFAIFEEIYTNEQDLAGVEKIDTLIQMGFILPEGYKLVAVRHCGKQNLVKENTLIFIAKTSFEVSI CRELKVKIListeria monocytogenes strain 1/2a 35152Monocin ORF 131 >antigen A, phage tail-like protein SEQ ID NO: 008MAFEENLYCDYTPGAAKAVAGKDVILAVFNAAGDKLLAVAGQQGLTVNRSKDSIEITSKDTVGGWKSKIGGMKEWSIENDGLYVADAESIAKELAKYFESDSPVCVKIINQASKKGLFGGLAIVADYSFEAPFDEAMTYSVKLDGMGALVDLTITEGGDQMPGETPVAPAEListeria monocytogenes strain 1/2a 35152Monocin ORF 132 >hypothetical protein SEQ ID NO: 009MFEVNDTTYILRFNKQKVKTVELTSGISLVAALTANKGILSYQVIETLFVSGLVEEKGLVPVKQKEALEIFDKLVEEQGLISLNVAVIEKLQEDMGFLFRListeria monocytogenes strain 1/2a 35152Monocin ORF 133 >hypothetical protein, phage-like SEQ ID NO: 010MNHDFDLEKQFAFFVVNFQMSKGDFEELTEVEKNFIMKEWENKVIFESTMLRNAVLNA EQNLNRKRNSRFIDLEIKKRQKKADVNYTVNALQAISDNEAKEGKAWIDRIYGANGLRRPKNKEERGKMNGGV Listeria monocyingenes strain 1/2a 35152Monocin ORF 134 >tape measure protein SEQ ID NO: 011MAESKSITFELNESVLTAQVGRLDEMAMVVERRFSELKMTIEDVGNADPGSKISESLGGLQSGLGTISSAFGQLGSSSEAITSGFGTAVGSVGGITDAFKNLGSSVQNGTLFSSLATGIGGMSTMLGGVSGGVQGITNLASGFMELKNHLGGLMSSIGGVGGIMGKLTSPMGLVIIGIVALVAAFTYLMTTNESFRNTVMSVVTQVAQLFGQLVASLMPIIIVIQIVTAVMQIGAALMPMVMQFISFFAQLLAQLMPFINMLISMLMPVIIVIQIVQVVMSLVSALLPSIIVITVIQGIIVISVIQFLIPIIIVIQIATVVVQIVVTIISYISKIMPIVMTIIGVIVSITTTIISYVVIIATTIASVIGKIISFIASVITAVIGIVQPIIAFITNIFTTIVTIIGAAFQMVFTVASKIWNSIIVISTISGIIDGIKAVITGISTTVSSVFNGVKRIITGVFDGIKSAWGGLTDFVGNIFDGVSSAIQTVVDNVKGFVNVVIRGINGAIGLINKIPGVEIGKIPQLISGTTNFQGGFARMNEGGRGEMVVLPSGSQVIPHDATMKYARESARGNKSMLYTSQGADLARVENLLERLLQKNPVIKMDDKVVAEVVSRNQANSFD QYNYTMGGAAYSListeria monocyingenes strain 1/2a 35152Monocin ORF 135 >phage tail component SEQ ID NO: 012MSDLFLELNGKVHSLSETFPGLSVQEVSRQSPQLSMETAEIAGTDGVIPGMTQFKPFIFSAKCNLQALDIPDYHLAVREIYEFLFQRDSYYIWSDQMPGIRYEVEEPKPVDFSRESDRVGLLTIEFDVFKGYAESRGTSLDPMTFEVDLWQMGMNLSNRDDLFYVFRENTFRVYNAGSDRVNPLIVIRRELDIAMTANGTPTIFINLTTGESFEYRKELQKTDVLLLNNIYPLVNNRRVGKDTNHGIITLEKGWNDFE1KGVTDVTIAFNFPFIYRListeria monocytogenes strain 1/2a 35152Monocin ORF 136 >phage tail protein SEQ ID NO: 013MDYVIIQSMDKEVEEILTDIDYGSFSYDYEKNTSRAISFTVNKTKQNAAIFDLVGNEAILTYQGQQFVIKKCTPKSIGGTISKQITAQHICYTVQDHVQYNVKSGRKKYSIQTVLEFALQDNVLGFSYEIQGSFPLVELEDLGNKNGLELVNLCLEEFGARYADNKKLYFYDEKSWYVRTEKQFRYLYNTEEVSVDTNTDNLKTEIKCYGKQKENADKLTGDNKYMAVVTYTSPNEAIYGKRMANAKSDDKITNNDDLLIFAKKQILDVPETALTIAYKGKEPVSERDVWYFMEPMGFETEVKVTKIKSSUMSKKFQEIGFSNSRRDMVRIQTQIANQVKKASVDTNKINSFSSIAMNAYDSRILTEVVGVVDGD Listeria nionocytogenes strain 1/2a 35152Monocin ORF 137 >hypothetical protein, phage-like SEQ ID NO: 014MATEIRVLKNVDDTVFYPKTHVTAVEGLDSATTTTSGLMPASDKTKLNGIEANAEKNNVTAIDIANWNKKQDAILVSENGSNFKITVTNAGELKATKVEListeria monocytogenes strain 1/2a 35152Monocin ORF 138 >hypothetical protein SEQ ID NO: 015MKLDLWKWEMLLQGREFRNKTNDNWQKLMDWSDFISTGLSAIYVYVNKADATLNNKIDTVDKAVNARVNELISGTEQLSEVVDARSDAFGARYPVLRERLNQEQLNFSKKSTIQFDASTIISMEKQDIGLLTSKKISEAQTVCFLNISSLDEEADIVLEKTGETSFSDNLTSLVFAKIGTNERYQMEPVGA Listeria monocytogenes strain 1/2a 35152Monocin ORE 139 >hypothetical protein SEQ ID NO: 016MTHKRMLQTKEDNSKEQFYPETHVAGIVGLTEYVSGQLPTGVVSVNGKAGRVLLDAEDVHAAKKSHTREVATYTTDGFMSSFDKQKIDQLVSPEAGVTSINGKTGIVDLFASDLDAAEINHTHAEATTTESGFLSIDDKEKLDAIQVIALETIKEVIEListeria monocytogenes strain 1/2a 35152Monocin ORF 140 >receptor binding protein SEQ ID NO: 017MTKIVKMSEKNEHGTLEQFYPETHAEAVKGLVSVSEEEKTIWDQKESTAGAEQKANTALNSAKDYVDTIGEGTVIFKGANLMGAGQSFKWDASKLKFGMTLLFSRYDAANNTPQDYYYHSVFLSKAQLVELAGKOLVQMPSTTYGDRKYLYVSTTGLSGEEFDNSNYAAWALR QVTIIVIListeria monocytogenes strain 1/2a 351.52 Monocin ORE 141 >holinSEQ ID NO: 018MEVILKFGILGFGAIFGYLFGEVDLLVKVLVCFWADYISGLLASGYLGELSSKMGFKGIAKKIAILILVAIAHQIDLILGTHNTTRDAVIFFYLANELISILENFVRMGMKVPEVLKNLILIFDAKSGEDEEKHDKDMD Listeria monorytogenes strain 1/2a 35152Monocin ORF 142 >lysin SEQ ID NO: 019MRKNMTKIWIDAGHGGKDSGASGNGLVEKNWVLTVAKQLQTELVKAGFEVGMTRTNDTFYELSDRAKKANSFKADLFISLHFNAGGGKGYEDYIYTSVPAATVEIQKIIHKNIITKVTKHGMNDRGMKKANFAVLRETAMDAILLEAGFCDSTDALILEKKAYQTDYCLGIVSAVQIFGAMVTKYRAGKYLTSDDAISGTNIKGYLEAGTKVFVYKETEKTLNLTTTKGVPGS WVLKTEVNTGKRListeria monocytogenes strain 1/2a 1144 (DuPont)A118 propliage tail fiber and downstream ORFs SEQ ID NO: 020atgacaaatcaaatctttaaatcagctattcttgatttttctgttagtgcacagaacgctaaagctaatgttcctcagataaaatttagtacgcaagactctggagggactgcgcgattaaagtttactgcaaaaaaagatgataacaatttaccactttcaagcgcggcagaggtaacgcttgctatggtattgtetgttggcaaaaaatacgaaagtagctacattgttaatccagaaataattaacagaacagaaggtgtttttgaatactcattgactgatgagcaaataagtcacgacggacaagctaatgcagaattgtacgttaaatatccaaatcaaacaatgcaaatcaatcgttttagttttgttattgaaaaagcgatgattgatgataattttttgcccgttgctacctattatgttgaaaaatgggatgattacgaaaaaatatttaacgaaaaagtggaaattcttcaaaatgaaattgatgatttgcaaggacaagctactgaattaaaaaacacattcgatagtettaatccagaccaatttccecaaaaagcagattttgaaaatcatataaaeaacacaaacattcatgtgacgatgactgataaaacaaattggaatacaaaagaaaatactgcgggatcacaagcaaaagcggatagtgcattaaactctgctaaagcatatacagatageaagatggatagttacggagettggataaatgtacccctcgcetctggttactcaactggcgacagtaatacacctcaatatcgactggtagcaaaacaaacattaccggtttgaaaacttttgctgaattccgcggatcagttgctggtacatttattagtacagcaaatagtactettgcaacaatgcccgctggcacaagaccaattgtcacttattacggtgctgccacttcaaataacgggaacggtggtcgtattgetattccagttgacggaaagctattacaagtgtcatctacagataatgctaatccttcgtacgtaagcctttcaacgatattatacgaagttggcaattaggaggagtaaacatgaactataaacagattacgcatatgatgaaaatggcaattatctcgaaacaatacttgtgtttgaagatgaaaaaggtttaatcaatcaccgaaaaattctacaaatattgaaccttccataatcgaaaacggcatagcaagagcaatgtattatccgcgtatgaatggggaagattgggacgaagaaaagaaaatatgggaattagaaaatccaatcataccccagaaaaaacggaaatagaaaaattaagagaggaattactactcacccaagaagcgttagcggeattgttcgaaagtaatttagggtgatgaaatggettatatgataccaatttacgtgaatttagtgatgaataatcgaaaaactattgaagaagttcctgegaatttgcgaggtcaggtaaaagcaaaagtggatgagctaaaacaagaacaacaacgaatacagtcagaagaaatagaagccgaataggcttatttatatggttgatgatgaaaatgtatgatggactaacaaaagttttgattatgctttagcgaaagaaattttcttcgcggcgctctttgtagcgctttttataatettactaattatcacaaaaagaatttgggatgattcgaaaattgtaagaatagaaatgaaagaagangegaaaaagtggaggaagaacgagagaagcgtaataaggaatcgaaagaagagagagataaatttataagtaegaataacgaacanagcgattgatgtataggcaaaatgacatgatgaaacagcaacaacaatcaattgacagettgtctaaatcagtcggaaagttagctcacaaagtagatttgatggaacacaaaataacgaatgaaggatgatagaaatggagtttggaaaagatttartagtttacatgacattatattagttgtaacacctgtgtttgttcagcgattaagaagaaggagttagtaccgtctaagtggcttccgactgttagcatacttattggtgctattctgggcgcattagcaacgttyttggacggctctggatcgcttgcaacgatgatttgggcaggcgattagcaggagctggtggtactggattatttganaatttactaatcgaagcaaaaaatatggagaggatgataaataaListeria monorytogenes strain 1/2a 1144 (DuPont)ORF 2345 >A118 tail fiber gene SEQ ID NO: 021MTNQIFKSAILDFSVSAQNAKANVPQIKFSTQDSGGTARLKFTAKKDDNNLPLSSAAEVTLAMVLSVGKKYESSYIVNPEIINRTEGVFEYSLTDEQISHDGQANAELYVKYPNQTMQINRFSFVIEKAMIDDNFLPVATYYVEKWDDYEKIFNEKVEILQNEIDDLQGQATELKNTFDSLNPDQFPQKADFENHINNTNIHVTMTDKTNWNTKENTAGSQAKADSALNSAKAYTDSKMDSYGAWINVPLASGYSTGDSNTPQYRLVAKQTSTGLKTFAEFRGSVAGTFISTAINTSTLATMPAGTRPIVTYYGAATSNNGNGGRIAIPVDGKLLQVSSTDNANPSYVSLSTILYEVGNListeria monocytogenes strain 1/2a 1144 (DuPont)ORF 2344 >A118 chaperone SEQ ID NO: 022MNYKQFYAYDENGNYIETILVFEDEKGL1NQPKNSTNIEPSHENGIARAMYYPRWNGEDWDEDKKRWELENPIIPAEKTEIEKLREELLLIQEALAALFESNLGListeria monocytogenes strain 1/2a 1144 (DuPont)ORF 2343 >A118 chaperone SEQ ID NO: 023MAYMIPIYVNLVMNNRKTIEEVPANLRGQVKAKVDELKQEQQRIQSEEIEAEListeria monocytogenes strain 1/2a 1144 (DuPont)ORF 2342 >A118 chaperone SEQ ID NO: 024MYDGLTKVFDYALAKEMFFAALFVALFIILLIITKRIWDDSKIVRIEMKEEREKVEEEREKRNKESKEERDKEISTMNEQQREMDRQNDNIMKQQQQSIDSLSKSVGKLABKVDELEHKI TKListeria monocytogenes strain 1/2a 1144 (DuPont) ORF 2341 >A118 holinSEQ ID NO: 025MEFGKELLVYMTFLVVVTPVFVQAMKTEINPSKWILPTVSILJGAILGALATFLOGSGSLATMIWAGALAGAGGTGLFEQFTNRSKKYGEDDKListeria monocytogenes strain 112a F6854GenBank: EAL07464.1 >receptor binding protein SEQ ID NO: 026MTKIVKMSEKNEHGFLEQFYPETHAEAVKCiLVSVIEEEKTTWGGKETTAGAEQKANAALNSAKDYVDTIGSGIVIFKGANLMGAGQAFKWDPDKLKFGMTLLFSRYDATTNTPQDYYYKSVFLSRAQLLEIAGGGVLIQMPSLTYGDKKYFYVSTTGISGHADNSNYKAWALR QVTIMListeria innocua strain 6a 33090Monocin ORF 174 >receptor binding protein SEQ ID NO: 027MTKIVKMSEKNEHGTLEQFYPETHAEAVKGLVSVTEEEKTTWNEKETTAGAEQKANTALNSAKEYVDTIGKGTBIFKGANIMGAGQKYTWSSSKLKFGITLLFSRYDSANNTPLDYYYHSVFLSKAQLAELAGKGLLVPMPSAIYGERKYLYVSETEVAGHNDNTNNASWALRQL TVMhyperspank_MCS_lacI cassette SEQ ID NO: 028GAATTcGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGCTCTAGCTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTGTTAACTCTAGAGCTGCCTGCCGCGTTTCGGTGATGAAGATCTTCCCGATGATTAATTAATTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATGCAGCAATGGCAAGAACGTTGctcgagggtAAaTGTGAGCaCTCACAATTcATTTTGCAAAAGTTGTTGACTTTATCTACAAGGTGTGGCATAATGTGTGtAATTGTGAGCGGATAACAATTAAGCTTagtcgacagctagccgCATGCAAGCTAATTCGGTGGAAACGAGGTCATCATTTCCTTCCGAAAAACGCTTGCATTTAAATCTTACATATGTAATACTTTCAAAGACTACATTTGTAAGATTTGATGTTTGAGTCGGCTGAAAGATCGTACGTACCAATTATTGTTTCGTGATTGTTCAAGCCATAACACTGTAGGGATAGTGGAAAGAGTGCTTCATCTGGTTACGATCAATCAAATATTCAAACGGAGGGAGACGATTTTGATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGITGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGIGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTICCCGACTGGAAAGCGGGCAGTGAListeria monocytogenes strain 1/2a 35152, Listeria monocytogenes strain 1/2a 1144(DuPont)Monocin ORF 140, A118 tail fiber gene >monocin-A118 phage tail receptor binding protein fusionSEQ ID NO: 029MTKIVKMSEKNEHGTLEQFYPETHAEAVKGLVSVSEEEKTNWNTKENTAGSQAKADSALNSAKAYTDSKMDSYGAWINVPIASGYSTGDSNTPQYRIVAKQTSIGEKTFAEFRGSVAGTFISTANSTLATMPAGTRP1VTYYGAATSNNGNGGRIAIPVDGKLEQVSSTDNANPSYVSLSTILYEVGN Bacteriophage TP901-1 >major tail protein SEQ ID NO: 030MAELTAKQGKDIILLYRVLSKASTEAAWKLAFQTEHSNEKTRDYNTTATKDGPVGALAEVEYSLSATSIAANGDPHLDEMDKAFDDAAIIEVWEIDKAEKATLGLDSGKYKAKYLRAYLTSFSYEPNSEDALELSLEFGVFGKPQKGYATLTTEQANVVQYVFKDTVRGTP901-1 like phage from Enterococcus avizon ATCC 14025 SEQ ID NO: 031MPEVTGFENQLYCDFSQSATKAVAGKNILLAIFNMTGDKLLAIAGQQGLTINRSKDSIEITSKDTKGGWKSKIGGMKEWSIDNDGLYVRDDESHKVLGQYFDGDDPVCIKVLDMQSKTGMFGGLAIVTDYSLEAPYDDAMTYSIKLDGMGALVDLSDSEANQMPEGTASIKLNKTTASIVVDANESLIATVQPSTDTVSWKTSDVTVATVDNTGRVTGKKVGNAIITATSTSKEV ATCLVTITASListeriophage A118 >major tail protein SEQ ID NO: 032MRIKNAKTKYSVAEIVAGAGEPDWKRLSKWITNVSDDGSDNTEEQGDYDGDGNEKTVVLGYSEAYTFEGTHDREDEAQNLIVAKRRTPENRSIMFKIEIPDTETAIGKATVSEIKGSAGGGDATEFPAFGCRIAYDETPTVTKP Listerlophage A118 >tape measureSEQ ID NO: 033MSDGSVVIEISLDDKKADKQLDAFEKDLAKAGTNAGAALDKAYREAVSDIASQSKRLKDTFVNAFKSMGNAGSNALKASLNFIRELPSNVQAALSKLASTVKTGFVNAAKASITAVKNLGTSIKNTAVNIKNGFFSIAKTVQSSIMSAVKISINVIKSIPSAIKSAGSSIKSALVSSLQAAKMAAISFAQTTVKVIKSIPGAAKTAATAVKNSFVVAYKAVVVAAYMSVKGTISAVKAIPSATKSAALAVSSAMKTAFSAVVSAAKTTGTTVKTALTNGFSAIKSGAKTAGQVGISALKGLGNAAKSTGSLIKNGLVSGFNAARSAAKGAGAGMREALKNSVEKPAEQARFSILRLAAAFGLIAATKNVVGSAIGRVDTIDTATKSLTVLTGSAKDAQLVMTDLTAAIDGTPIALDAVALGAKKMVAAGMQAANVKPVFTAIADAAYGVGNGSESIDQMTDAISALQASGVAYSDDINRLVDAGVPAWILANSTGKSVGEMKKYVSEGSLESTKAIAMLTKGIEEGTTGMAGNTAKMAGLAKTAGNTISGSFANMKTAAVKSLANIAENLKGPIIQALDVAKNAFKQFAAVTASPEFQKKLSDMIQKIKELIPVMVKLAPTILKVVSAMLALQAVSSVYVAFSNIGKMFVPLKNGLFVIATGFMKLAKTIREEPITAIKNLAFAIKYFIVTSGAVIAIVGAVIAVLYGMYAAFKENTANIKGFLSGMFDAVKNSFGKIVDVFKQIVSALKPVGSGFKDILKYIGVGVWVAFGIVLATVVDIIQVLARIVLVAIKGLQGLYYMKAAFQALSGDLKGAKKSLEQSKDAFVDAGSAIKDAFNKDNYALTGTIESLKEMGGEAEKTGTKAETSNKKISSSLKLVESTAKQTEATVTKSNQAIDTMLSGGVDQYGNKLSEKTKSFLNAAKELYGQYQESAKKSQDKYSVAMEKAQSLEGDKRKKAIADANATLVAEIDKNNGTLLTLQADYAKLLKDNKWVDGTELTAQQKKFLQQQTADIQAELAKQNQLYVEGNLLKLANGKTLNEKERATIEVQKSLYGDRKKAVEIGEKELADLKRKKSDATTETEKANYQIQIDEQTKKNKTLAGNLQKWASEMNAIIANGGTLNAETFAKGLSEMGNISDEQLGAVWQDFVKVSGSIDNTLAGLAAVMSQRGGEGVQAFVTALQSGDYTTAALKINDDVLNTISGLPNSMFLNGQSGKDQFLLAIKSGDFQGAGKFLLDGVKMGADPLPGEMEKNGKKSGDAQAKGVKSTAEANKSAGKEKNNAKSGAFDPNLFKMTGSKNSSGFNNGILGGKDGAFSAGTSVGGSAKSGAASVDSSGVGSDFAAGFANGIRSGAGAVGEAAASIAAKALAAVQKKQDSHSPSKKSKKLGGDFGSGYSLGIASKTKAVTKAASNLVAGALGTEKQIKKLSSTLKDKVSSAIDAGLHSKNKSRGQLKQAKALNSIEGYIAQQTNRLAATAKKRDKVVAQLKAANTKMADLTKQSKEYAASITEKMQSYGSISNVDAENPQSIQQEMQKRLKEIKAFQANVEKLRKKGVSKDIISDILESGVENGSSYAQALAKSDAKTIKAINSTQNQINSASKSMGNTAANAMYSAGINAAKGLINGLNSQKKQLEKTAKSIASTITNSVKKALKIHSPSRVAIELGKFFTGGLGNGVLAGAKGAVQSTNKMVDKVVNAASNMTVPTITLPKVSAEKALGLKSSDLNRTITVKAIVENESKNNSNSDLINAIEKSGGRPIILNVDGKVIADSTNNIALGNSTSLAFYGKGL Listeriophage A 118 >gp17SEQ ID NO: 034MATSLALVIEGKTYMLNELFDLEVGEVSREPPQIVNNYTEFAGSDGARTTDSNFSMFPISILCHFQTKTADLYHIKLDELLELIYQRKEYFLVHSKTPGKKYRVEEPSGVAIYRKAPGYADLTLEFDVFRGYSESLSSTLSDSEIDCDKWQFGQGLAMEDYRYTHTKSRFIYNGGSFDIDPREHQLTITIRGQNEGELVINNITTGDRFIYYPALSATDTLVIDSATPRINGNPCGRSTNHGLISLQKGENLIEISNTSHLDTRWDFSFLYK Listeriophage A118 >gp18 SEQ ID NO: 035MNSDIIVADFWKNNEEILTDFDKESFCETWTENEMWNIEFKVTQTNKNANCYSFLDYESSVFFGGQEFVVKQLSRDAVGKTLSKDIKAPHIYYTCQDGRQDDTITGSFTLEQCLTHIFKSDSRGFSWEIIDPSNILEKVQQENFGNNNYLTLIDQLLDDYGVVVIPDNREILVFKPRENYGAKTENFIRYKYNTDEASFDIDTLSLKTKIKGYGKVDSNGNNYFSPVTYTSPEAEKWGIRWQEPVSDERYTVVGNMQRRLKLELQDYPATTGSVILKNDYECEKGDYVLFIYEPLGIDYDVQIVAYKKYPFTIKAPEITLSNNKKSIVSIMAQLAKVLKGAKListeriophage A118 >gp 35 (regulatory)  SEQ ID NO: 036MNKTSYELKQEFPELNFVINNNLPTKLFGLIQNKVVIALEEPDLSENELRCTHEEAMEIWKYTAGDITKFNNVENIKQEKFARRKAHEYLVNIQSLALCYDLGYRTYYEAATFLNVTEKFLIEAVENYREKYGLMYNNGNYIIHFGSTIQVFQEDNSFYPYDYGCListeriophage A118 >gp61 (regulatory) SEQ ID NO: 037MSRKELRKKQWEVITMIEKSKTLTDRKNIIKKLETLEARGDKEKGLATPTQLLSITTVTEYRRLSKKLTDTEIAEDMGISRSALIEFKRKNGLSIRQKVATListeriophage A118 >gp66 (regulatory) SEQ ID NO: 038MGQLFNLPQVEDINYIQTVRAVRQFFKDYLTLRLMAGDRKFPNWITTMYKITPPNFSNEFHSKVEDAAIFINVDNVHAAQEAVKKYDAIMNQLEHIHRKILFEKFIFINLQDRTIMLDIPYEERQYKREKRKAVIELATTLGIEVLN

What is claimed is:
 1. An isolated nucleic acid molecule encoding an F-type bacteriocin, wherein the nucleic acid molecule comprises a first polynucleotide sequence that encodes the following structural proteins: a structural protein at least 90% identical to SEQ ID NO: 7; a structural protein at least 90% identical to SEQ ID NO: 8; a structural protein at least 90% identical to SEQ ID NO: 9; a structural protein at least 90% identical to SEQ ID NO: 10; a structural protein at least 90% identical to SEQ ID NO: 11; a structural protein at least 90% identical to SEQ ID NO: 12; a structural protein at least 90% identical to SEQ ID NO: 13; a structural protein at least 90% identical to SEQ ID NO: 14; a structural protein at least 90% identical to SEQ ID NO: 15; and a structural protein at least 90% identical to SEQ ID NO: 16, wherein the nucleic acid molecule further comprises a heterologous second polynucleotide sequence encoding a heterologous receptor binding protein (RBP), and wherein the RBP comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, and wherein the F-type bacteriocin has bactericidal activity against at least one strain of Listeria monocytogenes.
 2. A vector comprising the nucleic acid molecule of claim 1, wherein the nucleic acid molecule is operably linked to an inducible promoter.
 3. The vector of claim 2, wherein the promoter is a small molecule inducible promoter.
 4. An isolated nucleic acid molecule encoding an F-type bacteriocin of claim 1, wherein the nucleic acid molecule further comprises a heterologous promoter inducible by a small molecule, wherein the first polynucleotide sequence is operably linked to the heterologous promoter.
 5. The nucleic acid molecule of claim 4, wherein said nucleic acid molecule further comprises a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 5, and wherein the promoter is placed at 11, 14, 17, 20, or 23 nucleotides upstream of the nucleic acid sequence encoding SEQ ID NO:
 5. 6. A vector comprising the nucleic acid molecule of claim
 5. 7. An isolated F-type bacteriocin producer cell comprising the vector of claim
 2. 8. The isolated nucleic acid molecule of claim 1, wherein the RBP comprises the amino acid sequence of SEQ ID NO:
 29. 9. An isolated F-type bacteriocin producer cell comprising a first foreign polynucleotide sequence that encodes the following structural proteins: a structural protein at least 90% identical to SEQ ID NO: 7; a structural protein at least 90% identical to SEQ ID NO: 8; a structural protein at least 90% identical to SEQ ID NO: 9; a structural protein at least 90% identical to SEQ ID NO: 10; a structural protein at least 90% identical to SEQ ID NO: 11; a structural protein at least 90% identical to SEQ ID NO: 12; a structural protein at least 90% identical to SEQ ID NO: 13; a structural protein at least 90% identical to SEQ ID NO: 14; a structural protein at least 90% identical to SEQ ID NO: 15; and a structural protein at least 90% identical to SEQ ID NO: 16, and wherein said producer cell further comprises a second foreign polynucleotide sequence encoding a heterologous receptor binding protein (RBP), and wherein the RBP comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, and wherein the F-type bacteriocin has bactericidal activity against at least one strain of Listeria monocytogenes.
 10. The isolated producer cell of claim 9, wherein the first and second foreign polynucleotide sequences are contained within separate nucleic acid molecules.
 11. The isolated producer cell of claim 9, wherein the first and second foreign polynucleotides are contained within one nucleic acid molecule.
 12. The isolated producer cell of claim 9, wherein said first and second foreign polynucleotide sequences are operably linked to a heterologous promoter inducible by a small molecule.
 13. A method of producing an F-type bacteriocin, comprising exposing the F-type bacteriocin producer cell of claim 12 to an inducing agent in a concentration effective to induce expression of the F-type bacteriocin via the inducible promoter, thereby producing the F-type bacteriocin.
 14. The isolated producer cell of claim 9, wherein the RBP comprises the amino acid sequence of SEQ ID NO:
 29. 15. An F-type bacteriocin, wherein said F-type bacteriocin is encoded by a nucleic acid molecule comprising a first polynucleotide sequence and a second polynucleotide sequence, wherein said first polynucleotide sequence encodes the following structural proteins: a structural protein at least 90% identical to SEQ ID NO: 7; a structural protein at least 90% identical to SEQ ID NO: 8; a structural protein at least 90% identical to SEQ ID NO: 9; a structural protein at least 90% identical to SEQ ID NO: 10; a structural protein at least 90% identical to SEQ ID NO: 11; a structural protein at least 90% identical to SEQ ID NO: 12; a structural protein at least 90% identical to SEQ ID NO: 13; a structural protein at least 90% identical to SEQ ID NO: 14; a structural protein at least 90% identical to SEQ ID NO: 15; and a structural protein at least 90% identical to SEQ ID NO: 16, and wherein said second polynucleotide sequence encodes a heterologous receptor binding protein (RBP), and wherein the RBP comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, and wherein the F-type bacteriocin has bactericidal specificity against at least one strain of Listeria monocytogenes.
 16. A method of killing a Listeria monocytogenes, comprising contacting the L. monocytogenes with an effective amount of the F-type bacteriocin of claim 15, whereby the F-type bacteriocin binds and kills the L. monocytogenes.
 17. The method of claim 16, wherein the contacting is with a surface contaminated with L. monocytogenes.
 18. The method of claim 17, wherein the contacting and killing are at 4°-10° C.
 19. The F-type bacteriocin of claim 15, wherein the RBP comprises the amino acid sequence of SEQ ID NO:
 29. 20. An isolated nucleic acid molecule encoding an F-type bacteriocin, wherein the nucleic acid molecule comprises a polynucleotide sequence that encodes the following proteins: a structural protein at least 90% identical to SEQ ID NO: 7; a structural protein at least 90% identical to SEQ ID NO: 8; a structural protein at least 90% identical to SEQ ID NO: 9; a structural protein at least 90% identical to SEQ ID NO: 10; a structural protein at least 90% identical to SEQ ID NO: 11; a structural protein at least 90% identical to SEQ ID NO: 12; a structural protein at least 90% identical to SEQ ID NO: 13; a structural protein at least 90% identical to SEQ ID NO: 14; a structural protein at least 90% identical to SEQ ID NO: 15; a structural protein at least 90% identical to SEQ ID NO: 16; and a protein at least 90% identical to SEQ ID NO: 17, wherein said polynucleotide sequence is operably linked to a heterologous inducible promoter.
 21. A vector comprising the nucleic acid molecule of claim
 20. 22. The nucleic acid molecule of claim 20, wherein the promoter is a small molecule inducible promoter.
 23. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule further comprises a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 5, and wherein the promoter is placed at 11, 14, 17, 20, or 23 nucleotides upstream of the nucleic acid sequence encoding SEQ ID NO:
 5. 24. A vector comprising the nucleic acid molecule of claim
 23. 25. An isolated F-type bacteriocin producer cell comprising the nucleic acid molecule of claim
 20. 26. An isolated F-type bacteriocin producer cell comprising the nucleic acid molecule of claim
 22. 27. A method of producing an F-type bacteriocin, comprising exposing the F-type bacteriocin producer cell of claim 25 to an inducing agent in a concentration effective to induce expression of the F-type bacteriocin via the inducible promoter, thereby producing the F-type bacteriocin. 